Thèse 2022upasf035

De Doctorat

Keywords: Métalloenzymes, métalloenzymes artificielles, enzyme chimérique, réactions en cascade Metalloenzymes, artificial metalloenzymes, chimeric protein, cascade reactions

Titre : Enzymes chimériques pour la catalyse énantiosélective de réactions en cascade.

In the context of sustainable green chemistry, enzyme-catalyzed chemical reactions represent the best eco-friendly scenario in organic synthesis. Enzymes can be used under eco-friendly conditions (in aqueous solution, at room temperature, under atmospheric pressure,) to obtain specific chiral products and avoid the formation of undesirable side-products. [START_REF] Schmid | Industrial biocatalysis today and tomorrow[END_REF][2][START_REF] Di Meo | αRep A3: A Versatile Artificial Scaffold for Metalloenzyme Design[END_REF] Natural enzymes are classified as the most powerful catalysts of natural organisms.

They can catalyze efficiently different kinds of reactions. They have evolved for millions of years to reach efficient catalysis for the transformation of very specific substrates which at the same time limits their applications when using non-natural substrates. Additionally, enzymes can catalyze only limited types of reactions and desired product stereoisomers are not always accessible. [START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF] To overcome these major limitations of enzymes, the field of artificial metalloenzymes has evolved. [START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF] In the context of ecofriendly chemistry, artificial metalloenzymes result from the incorporation of a catalytically competent metal cofactor into a protein scaffold. [START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF] The construction of these hybrid biocatalysts combining biological components and synthetic chemical components include genetic mutation, chemical modification and/or combination of these two methods. [START_REF] Matsuo | Artificial enzymes with protein scaffolds: Structural design and modification[END_REF] Hybrid biocatalysts have several advantages such as: broad range of substrates and reactions, possibility of working in a chiral environment provided by the residue of the protein under mild operating conditions. [START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF] With the fast advances of artificial metalloenzyme's research, as well as their advantages, they have the potential to become legitimate contenders to natural enzymes for their use in a wide range of fields, from chemical and food industries to diagnostics and medicine. [START_REF] Kuah | Enzyme Mimics: Advances and Applications[END_REF] Thus, artificial metalloenzymes are categorized in the production of fuels, in biological reactions (Ribonuclease A mimics, ATPase mimics) and in green and sustainable chemistry (Diels Alder, Suzuki-Miyaura and others for chemical synthesis). [START_REF] Kuah | Enzyme Mimics: Advances and Applications[END_REF] Different strategies were described over the years to construct artificial metalloenzymes. These strategies will be discussed in chapter 1, section 1.1.1.

But in this project, a novel strategy seemed most appealing to us: the construction of chimeric enzymes. Introduction

A.2. Chimeric enzymes

Over the previous decade, many fields like biochemistry, protein chemistry, molecular cloning, random and site-directed mutagenesis, directed evolution of biocatalysts… have all made it possible to use a wide range of enzymes and microbial cultures as tools for organic synthesis. [START_REF] Patel | 9.12 Industrial Applications of Biocatalytic Hydrolysis (Esters, Amides, Epoxides, Nitriles) and Biocatalytic Dynamic Kinetic Resolution[END_REF] The utilization of various classes of enzymes for the catalysis of a wide range of chemical reactions can result in a wide range of chiral molecules. [START_REF] Patel | 9.12 Industrial Applications of Biocatalytic Hydrolysis (Esters, Amides, Epoxides, Nitriles) and Biocatalytic Dynamic Kinetic Resolution[END_REF] Fusion proteins, also known as chimeric proteins, are produced from the translation of a gene that combines two or more genes that previously coded for different or similar proteins. Indeed, when this fusion gene is translated, it produces a single polypeptide with functional features that are derived from those of the original proteins. [START_REF] Patel | 9.12 Industrial Applications of Biocatalytic Hydrolysis (Esters, Amides, Epoxides, Nitriles) and Biocatalytic Dynamic Kinetic Resolution[END_REF] Furthermore, chimeric proteins are advantageous because they reduce costs by replacing multiple enzymes by a single enzyme able to catalyze two or more reactions. Hence, they are advantageous in atom economy which is the second of the 12 green chemistry principles that aims to optimize the incorporation of all the atoms of the starting materials into the desired final product without generating side products. [START_REF] Strohl | Chimeric Genes,Proteins[END_REF][START_REF] Harper | Expression and Purification of GST Fusion Proteins[END_REF][START_REF] Li | Green chemistry for chemical synthesis[END_REF] Introduction

B. AIM OF THE PROJECT

In the context of the preparation of artificial enzymes for applications in chemical synthesis and inspired by natural and artificial enzymatic catalysis, the ambitious challenge of this project is to fuse structurally related enzymes possessing different but complementary activities to catalyze different reactions and reactions in cascade.

We aim to prepare artificial chimeric metalloenzymes that possess multiple active sites by the fusion of catalytic domains and the deletion of non-catalytic domains of two related enzymes. This chimeric enzyme will be used to catalyze reactions in cascade with maximized atom economy under ecofriendly conditions (scheme 1).

Scheme 1: Preparation of artificial chimeric metalloenzyme containing multiple active sites.

M=Metals

To prepare the chimeric enzyme, we chose to rely on the cupin superfamily of proteins.

Cupins are proteins characterized by a structure formed of a barrel of four β sheets containing the active site. [START_REF] Dunwell | Cupins: the most functionally diverse protein superfamily?[END_REF] However, artificial metalloenzymes comprising an active site formed by β sheets are rare and to understand the effect of active sites formed by β sheets on catalysis, it is Introduction thus necessary to transform other cupins into artificial metalloenzymes and to study their activity. [START_REF] Nanda | Designing artificial enzymes by intuition and computation[END_REF] In this project, we selected two related enzymes from the bicupin family: Mannose-6-Phosphate Isomerase (PMI) (Figure 1A) and Pirin Like protein (PLP) (Figure 1B) are similar bicupins characterized by a structure formed of two β barrel domains, each possessing one active site in one of their two β-barrel domains. So, they are formed only of two domains composed each of one barrel of four β-sheets and connected by a single inter-domain consisting of α helixes. [START_REF] Ahmad | Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-D -arabinonhydrazide[END_REF][START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] Figure 1: A) 3D structure of PMI B) Swiss model of PLP

The bicupin chimera PMI-PLP (Figure 2) will therefore be fused at the inter-domains of the two original bicupins. Recently in our laboratory, the crystal structure of mannose-6-phosphate isomerase of Candida albicans was obtained. [START_REF] Ahmad | Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-D -arabinonhydrazide[END_REF] Aiming at the preparation of artificial enzymes, we substituted the Zn(II) cation of its active site by a Cu(II) cation. This substitution transformed the isomerase into an artificial metalloprotein.

PLP is present in Pseudomonas stutzeri zobelli and it is thought to contain one atom of iron in its active center. Its biological role remains unclear. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] Its structure resembles that of the PMI. PLP has also been transformed into an artificial enzyme via the substitution of its Fe(III) cation by a Cu(II) cation, which provided it with quercetinase activity. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] The PMI active site is found in the β-sheet barrel domain of the C-terminal side of PMI, while the PLP active site is located in the β-sheet barrel domain of the Nterminal side of PLP. The superimposition of both starting enzymes has shown that the zinc-containing PMI's active center aligns with the empty cupin domain of PLP, while the iron-containing PLP 's active center aligns with the empty cupin domain of PMI (Figure 3). Introduction Thus, we plan to design a functional chimeric enzyme by combining the catalytic domains of PMI and PLP (C-terminal side of PMI containing the active site and the Nterminal side of PLP also containing its active site), which should avoid denaturation of the produced protein and should lead to a correct folding.

B.2. The strategy to produce the Chimeric enzyme

The strategy we followed to prepare the PMI-PLP bicupin chimera started first by structural modeling studies that allowed us to identify the linking point. We used elNémo to simulate the normal modes of vibration of all the atoms of both PMI and PLP, except hydrogen atoms, starting from the lowest frequency. We studied the movements of all atoms/domains in PMI and PLP and we visualized their movements.

Molecular biology techniques were then used to design the plasmid for the production of the protein (scheme 3). Enzymes are proteins folded into three-dimensional structures in most of which hydrophobic cores are surrounded by hydrophilic shells. This fold allows reactions to occur inside the hydrophobic portions, which gives the specificity of enzymes. [START_REF] Kuah | Enzyme Mimics: Advances and Applications[END_REF] In most cases, enzymes require the use of co-substrates i.e. cofactors which often causes an increase in the cost of production. [START_REF] Kuah | Enzyme Mimics: Advances and Applications[END_REF] As mentioned in the introduction, enzymes can catalyze only limited types of reactions and desired stereoisomers are not always accessible. To overcome these hurdles, artificial metalloenzymes have been developed. [START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF] Artificial metalloenzymes result from the incorporation of a catalytically competent metal cofactor into a protein scaffold (Scheme 4). The construction of these hybrid catalysts includes genetic mutations, chemical modifications and/or combination of these two methods. [START_REF] Matsuo | Artificial enzymes with protein scaffolds: Structural design and modification[END_REF] These powerful catalysts are conveyed with extraordinary catalytic properties provided by the non-native metal cofactor and with a protective and stereoselective environment provided by the hosting protein. [START_REF] Ghattas | Receptor-Based Artificial Metalloenzymes on Living Human Cells[END_REF] Scheme 4: Preparation of artificial enzyme by incorporating synthetic transition metal catalysts into natural protein.

Highly sophisticated catalysts can be chemically synthesized and can act like enzyme models that can catalyze desired reactions. Such catalysts -namely biomimetic catalysts-can be designed to mimic the active site of natural metalloenzymes. Their preparation however requires environmentally unfriendly protocols. Since chemically synthesized catalysts usually work in organic solvents, artificial metalloenzymes were Chapter 1: State of the art designed to overcome the limitations of both natural enzymes and chemically synthesized models (see Table 1). [START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF][START_REF] Lin | Rational design of metalloenzymes: From single to multiple active sites[END_REF] A chiral environment can be easily provided by biomolecule hosts under mild conditions in aqueous solution and which can host simple metal complexes to catalyze desired reaction under enzyme-like conditions. [START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF] Artificial metalloenzymes have thus several advantages such as: they can perform asymmetric catalysis to obtain pure enantiomers, they can catalyze various reactions, using a broad range of substrates, and can allow to obtain large scale products for a practical use while working under eco-compatible conditions. 4 -

Covalent attachment

This strategy requires the presence of a reactive group (such as maleimide) on the metal complex that will interact with a specific amino acid of the protein such as the SH chemical function of cysteine (Figure 4). Kaiser et al. were the first to demonstrate that novel catalytic activity can be created by covalent modifications of an amino acid residue with appropriately modified metal complex. [START_REF] Di Meo | αRep A3: A Versatile Artificial Scaffold for Metalloenzyme Design[END_REF][START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF] -Supramolecular anchoring

This strategy relies on the covalent attachment of the metal complex moiety to an inhibitor or substrate analogue that possesses high affinity for the hosting protein, and that is then used as a "Trojan horse" to drive it inside the active site (Figure 4). This method was historically introduced by Whitesides et al. [START_REF] Wilson | Conversion of a protein to a homogeneous asymmetric hydrogenation catalyst by site-specific modification with a diphosphinerhodium(I) moiety[END_REF] and a variety of enantioselective transformations were performed with such artificial enzymes, including sulfoxidation, epoxidation, reduction and Diels-Alder cycloaddition reactions. [START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF] -

Metal exchange

This strategy relies on the intrinsic affinity of a protein for a metal cation (Figure 4).

Kaiser et al. were the first to modify a protein by this strategy to afford an artificial metalloenzyme with novel catalytic properties by substitution of zinc by copper in carboxypeptidase A (CPA). [START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF] A separate bioconjugation step for introducing a synthetic ligand is not necessary. This strategy is nevertheless challenging, because the design must be so "perfect" that the protein binds the respective transition metal solely at the envisioned site. If alternative binding sites in the protein compete for the metal, then an undesired mixture of catalysts will be formed. In the worst case, some of the catalytically active metal salt will not bind at all, leading to catalysis outside the protein active site thus racemic products. [START_REF] Reetz | Artificial Metalloenzymes as Catalysts in Stereoselective Diels-Alder Reactions[END_REF] Chapter 1: State of the art

-Directed evolution

Directed evolution is a protein engineering technique increasingly used to improve enzymatic activity for different types of substrates and to enhance their enantioselectivity. [START_REF] Minard | L'évolution dirigée des protéines[END_REF] It also allows for non-biological activity to be conferred on an existing protein or enzyme. Thus, directed evolution induce an artificial activity and can help improve the activity by increasing the conversion and selectivity of the chosen reaction. In a first round of mutation, the gene encoding for the enzyme will be randomly mutated to obtain a library of mutant genes by random PCR mutagenesis.

Then, the mutant genes are expressed in microorganisms to produce various mutated enzymes. The catalytic activity of these mutants for the target reaction is then screened and the mutants with activity are selected (Figure 5). A new random mutagenesis step is performed on the mutated genes that provide enzymes the best activity to build a new library. [START_REF] Arnold | Directed Evolution: Bringing New Chemistry to Life[END_REF] Chapter 1: State of the art and applied it to hemoproteins in order to make them capable of catalyzing new nonbiological activities such as nitrene and carbene transfer reactions. [START_REF] Arnold | Directed Evolution: Bringing New Chemistry to Life[END_REF] -Fusion of multiple proteins

The formation of chimeric genes can result in proteins with modified functions and sometimes entirely new functions. [START_REF] Strohl | Chimeric Genes,Proteins[END_REF] In some instances, these chimeric gene products are selectively favored and then rapidly spread through populations. [START_REF] Bonde | Chimeric Genes[END_REF] Chimeric proteins can be prepared in vitro by fusing the structural genes of desired proteins into a suitable expression vector. The 3 'translational term of the first gene is deleted, as is the promoter of the 5' term of the second structural gene. The two genes are linked in the frame and expressed in an appropriate host (scheme 5). [START_REF] Bonde | Chimeric Genes[END_REF] In this project we were interested in this strategy to prepare an artificial chimeric metalloenzyme that possess a double active site by the fusion of the catalytic domains and the deletion of non-catalytic domains of two related enzymes. The development of such multifunctional chimeras is beneficial as not only these contain the same number of atoms as the starting enzymes do, but, in addition they can catalyze two or more reactions. 8

Some examples of reactions catalyzed by artificial metalloenzymes

Many artificial metalloenzymes prepared by the strategies described above have been reported in literature, in particular by Ward et al. [START_REF] Davis | Artificial Metalloenzymes: Challenges and Opportunities[END_REF] They have been reported to catalyze a large variety of reactions such as: [START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF][START_REF] Mahy | Current Applications of Artificial Metalloenzymes and Future Developments[END_REF] Redox reactions, Hydrogenation, ketone reduction, alcohol oxidation, epoxidation, dihydroxylation, peroxidation, hydrolysis, transamination, DNA-cleavage, C-C bond forming artificial metalloenzymes, Diels-Alder cycloaddition, hydroformylation, suzuki coupling, etc… Among the reported artificial metalloenzymes, an artificial Diels-Alderase was developed recently in the host laboratory from 1-amino-cyclopropane oxidase (ACCO), which belongs to the cupin family of protein.

ACCO is a non heme iron containing enzyme (Figure 6) that catalyzes the final step of the ethylene biosynthesis in plants. [START_REF] Bakkali-Tahéri | Characterization of Cu(II)-reconstituted ACC Oxidase using experimental and theoretical approaches[END_REF] In 2013, Thierry Tron et al. characterized for the first time a Cu 2+ reconstituted ACCO using experimental and theoretical approaches.

They have shown that Cu 2+ coordinated to the protein with a good affinity and it bound two histidines and two water molecules. [START_REF] Bakkali-Tahéri | Characterization of Cu(II)-reconstituted ACC Oxidase using experimental and theoretical approaches[END_REF] Chapter 1: State of the art ACCO was transformed into a Diels-Alderase by exchanging the iron(II) cation of its active site by a copper(II) cation. [START_REF] Ghattas | Cu II -Containing 1-Aminocyclopropane Carboxylic Acid Oxidase Is an Efficient Stereospecific Diels-Alderase[END_REF] The artificial enzyme obtained was more effective and more selective than any other known artificial Diels Alderase. The Cu(II)-ACC artificial enzyme could catalyze the cycloaddition of 40 equivalents of 2-azachalcone with an excess of cyclopentadiene and formed exclusively the (1R,2S,3S,4S) endo product with 100 % enantiomeric excess (scheme 6).

Scheme 6: Diels-Alder cycloaddition of cyclopentadiene and 2-azachalcone.

CHIMERIC ENZYMES

According to Greek etymology, the term "chimera" originates from the "Chimaera" a monstrous creature with the head of a lion, body of goat, and tail of serpent.

According to National Cancer Institute (NCI) dictionary of cancer terms, ''Chimeric'' means having parts of different origins. In medicine, it refers to a person, organ, or tissue. This may happen because of a mutation that occurs during development, or results of a transplant of cells, organs, or tissues from another person or from a different species. In the laboratory, a chimeric protein can be made by combining two different genes. The formation of chimeric genes can result in proteins with modified functions and sometimes entirely new functions. In some cases, chimeric proteins do not fold in the same way as the parental proteins. [START_REF] Strohl | Chimeric Genes,Proteins[END_REF] As mentioned before, the development of multifunctional chimera is beneficial and has many advantages especially in atom economy. [START_REF] Strohl | Chimeric Genes,Proteins[END_REF][START_REF] Li | Green chemistry for chemical synthesis[END_REF] However, it is important to engineer a chimeric protein with improved catalytic efficiency to generate bifunctional or multifunctional enzymes and to enhance the consecutive enzyme reaction rate. [START_REF] Strohl | Chimeric Genes,Proteins[END_REF][START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Fusion proteins can be obtained by different strategies mimicking the nature's strategy such as: tandem fusion, domain insertion and post translational conjugation. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Starting from partner proteins with similar working temperatures and pH profiles appear to be especially important for designing fusion enzymes, as mismatched parameters could affect overall bioactivity. For fusion enzymes, the tandem fusion approach is commonly utilized, and suitable linker peptides should be chosen to avoid severe interference between fusion partners. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] The orientation in which the fusion partners are positioned has a significant impact on their performance. The transfer of substrates, cofactors, and electrons can be considerably facilitated by a favorable orientation and distance between the active sites. When constructing fusion enzymes, it is always a good idea to take into consideration the known tertiary structures of each protein, the domain order on the polypeptide chain, and the length of linkers if any are used. This should favor the good folding of the design before it is built physically. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Many fusion enzymes were important in the biotechnology and biopharmaceutical fields. For example, pyrroline-5-carboxylate synthase and acetyl-coA carboxylase have been characterized as efficient biocatalysts in metabolic pathways; green fluorescent protein (GFP) is considered as a biosensor for monitoring signaling molecules. Chimeric genes are essential evolutionary forces because they provide a source of new genes, allowing organisms to create novel phenotypes and adapt to their environment. [START_REF] Bonde | Chimeric Genes[END_REF] Chimeric genes, as opposed to direct gene duplication, can result in proteins with altered and sometimes altogether novel functions. One of the most well-known chimeric protein genes has been identified in Drosophila and has been named Chapter 1: State of the art jingwei. [START_REF] Bonde | Chimeric Genes[END_REF][START_REF] Wang | The Origin of the Jingwei Gene and the Complex Modular Structure of Its Parental Gene, Yellow Emperor, in Drosophila melanogaster[END_REF] Its chimerical gene sequence contained a retro-sequence of alcohol dehydrogenase (ADH) and a non ADH parental gene sequence. [START_REF] Wang | The Origin of the Jingwei Gene and the Complex Modular Structure of Its Parental Gene, Yellow Emperor, in Drosophila melanogaster[END_REF] Biochemical analysis has revealed that the jingwei-encoded protein evolved a new functional role in the metabolism of the hormone. [START_REF] Bonde | Chimeric Genes[END_REF] Chimeric proteins can be also prepared in vitro by fusing the structural genes of the proteins in question into a suitable expression vector. Fusion proteins are divided into two categories: Type 1 and type 2 proteins (Figure 7).

In type 1, the 3 'translational term of the first gene is deleted, as is the promoter of the 5' term of the second structural gene. The two genes are linked in the frame and expressed in an appropriate host. Plant, mammal, bacteria and insect cells have been used as hosts but the most commonly used hosts are Escherichia coli strains. [START_REF] Strohl | Chimeric Genes,Proteins[END_REF] After transcription and translation, the cell will produce a single chain of polypeptide with the properties from both original genetic products. Fusion can be performed at one or both ends of a protein. Thus, it should be assayed whether one end or the other is more favorable for the sought-after biological activity. [START_REF] Bonde | Chimeric Genes[END_REF] In type 2, the sequences of two related proteins are interlaced to create a distinct protein, generally with a novel activity. Unlike the first type of fusion protein, which has two types of activity that are not novel, this type of fusion protein usually only has one type of activity that is however novel. 8 The growing number of sequenced genes and genomes, together with the use of polymerase chain reaction (PCR) to recover intact genes, open up a plethora of possibilities for structural gene fusion from many sources. The nearly infinite number of conceivable fusion partner combinations has made this technology extremely adaptable in many fields of biochemistry and biotechnology. [START_REF] Strohl | Chimeric Genes,Proteins[END_REF] Chapter 1: State of the art Genetic engineering has made it possible to create chimeric proteins via the fusion of genes encoding separate enzymes into a chimeric gene coding for a single multifunctional polypeptide. [START_REF] Bonde | Chimeric Genes[END_REF] For example, fatty acid synthases and the enzymes involved in aromatic amino acid biosynthesis exist as discrete enzymes in many plants and in most prokaryotic organisms. In other species, various combinations of multifunctional enzymes are present. Thus, they are considered to be examples of enzymes produced through an evolution process involving gene fusion. 24

Choice of the linker

The main problem in the production of chimeric proteins is the retention of activity following fusion. Hence, it's crucial to find the suitable fusion point to retain the original folding and thereafter the original activity. [START_REF] Bonde | Chimeric Genes[END_REF] The capacity of individual units to fold independently of the rest of the polypeptide chain after fusion is critical to the design of a chimeric protein. Misfolding otherwise often results in inactive structures. When developing and examining a linker region, there are numerous factors to consider. The first criterion is to avoid proteolytic cleavage within the linker, which restricts the linker's amino acid composition. [START_REF] Bonde | Chimeric Genes[END_REF] Linkers are found in all naturally occurring multidomain proteins, and their role is to maintain the required distance between two protein domains to avoid steric hindrance and/or to allow for favorable domain-domain interactions. As a result of this observation, researchers have used numerous sorts of naturally occurring linkers in their synthetic fusion structures. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] For example, in many recombinant therapeutic proteins, such as tailored antibody constructions, the immunoglobulin hinge region serves as a linker. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF][START_REF] Wu | Simultaneous targeting of multiple disease mediators by a dualvariable-domain immunoglobulin[END_REF] Natural enzyme linker sequences are also attractive possibilities for joining fusion partners: O-glycosylation, proline-and hydroxylamine-rich cellulase and xylanase linkers maintain an extended shape and are protected from proteolysis. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Fusion protein linkers have lately been studied and explored in terms of their features, functions, and instances. In general, various linker qualities should be carefully considered: length, amino acid composition, hydrophobicity, protease sensitivity, secondary structure, and probable fusion partner interaction. The linkers appear to be a critical component of a good fusion structure. Furthermore, because each sort of linker has its own unique properties, it's difficult to assume that a linker that works in one circumstance would work in another. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Researchers have developed a variety of artificial linkers in addition to natural linkers, which can be categorized into flexible, rigid, and in vivo cleavable linkers. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Chapter 1: State of the art (Gly)n and (Gly-Gly-Gly-Gly-Gly-Ser)n are the most commonly used flexible linker sequences in which the linker length is controlled by the copy number "n." [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] The high homologous repeats in this family of linkers' DNA encoding sequences, may limit protein expression levels and necessitate suitable codon pair selection. Rigid linkers may be desirable when sufficient separation of protein domains is desirable. A helixforming linker with the (EAAAK)n motif, for example, was created for this purpose. The intrinsic Glu-Lys+ salt bridges in this linker stabilize it, resulting in a rigid and prolonged shape. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] When the recombinant fusion protein is subjected to reducing agents or proteases, cleavable linkers are used to liberate free functional domains in vivo. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] In natural chimeric proteins, bulky amino acids as well as hydrophobic residues are avoided in linkers, excepted the smaller hydrophobic residues, alanine and proline. On the contrary, Glycine, serine and threonine are the preferred amino acids in natural linkers whereas other common residues such as asparagine, glutamine and lysine can also be found. [START_REF] Bonde | Chimeric Genes[END_REF] It can be useful to arrange the catalytic centers of various enzymes in a close vicinity, to increase the overall kinetics and yield of an enzymatic process, especially if it is based on successive stages. In this way, the intermediate product can be efficiently channeled to the second active site of the fusion protein. In nature, this has been achieved through the evolution of multifunctional proteins and multienzyme complexes with small linkers. [START_REF] Bonde | Chimeric Genes[END_REF] Interesting bioinformatics tools have been created to facilitate the logical design and selection of linkers. LINKER, an online application for automatically creating linker sequences for fusion proteins, was created. It should be noted that the server page is no longer accessible, most likely owing to a lack of updates. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Similarly, in 2002, R. A. Bank (PDB), as well as empirical recombinant fusion proteins reported in the literature and patents. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Chapter 1: State of the art

Design of chimeric enzymes

The structure's prediction is an important factor to be taken into consideration before proceeding to the construction and the expression of a chimeric enzyme.

Structure's prediction:

Although significant progress has been made in the development of synthetic fusion proteins, the structures of these proteins are difficult to predict. This is mostly due to their vast size and inherent complexity and flexibility. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] As a result, only a few structural features of synthetic fusion proteins, such as inter-domain interactions, have been well elucidated thus far. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] The interactions between domain pairs, can be crucial in the design of fusion proteins that need proximal domain placement for quicker electron transfer, substrate channeling, or cofactor regeneration. As a result, a greater understanding of the structure of the fusion protein could potentially speed up its rational design and increase its success rate. In comparison to experimental determination, computational protein structure prediction can be regarded as a costeffective option. Significant progress has been achieved in this field to better understand the structure of fusion proteins by utilizing individual domain structural information either from known structural data or from homology modeling. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] -Fusion Protein MODeller (FPMOD) is a modeling tool developed by Chiang et al. [START_REF] Chiang | A Modeling Tool for Sampling the Conformational Space of Fusion Proteins[END_REF] in 2006 that can construct fusion protein models using the structures of the fusion partners. The realistic models can be constructed by designating regions of flexible linkers and then rotating rigid-body domains around them to build random structures. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] -Rosetta software is used in computational modeling to analyze protein structures, to anticipate the structure of designed domain insertion proteins from scratch, and to create such fusion structures. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] -MODELLER is a program that is used to model protein three-dimensional structures in terms of homology or comparison. [START_REF] Webb | Comparative Protein Structure Modeling Using MODELLER[END_REF][START_REF] Martí-Renom | Comparative Protein Structure Modeling of Genes and Genomes[END_REF] It automatically calculates a model comprising all non-hydrogen atoms when the user aligns a sequence to be modeled with known related structures. MODELLER performs de novo modeling of loops in protein structures, optimization of various protein structure models with respect to a flexibly defined objective function, multiple alignment of protein Chapter 1: State of the art sequences and/or structures, clustering, searching of sequence databases, comparison of protein structures, and many other tasks. [START_REF] Fiser | Modeling of loops in protein structures[END_REF] -I-TASSER is a hierarchical approach to automated protein structure prediction and structure-based function annotation. Multiple threading alignment techniques are used in I-TASSER to recognize structural templates from the PDB. Iterative fragment assembly simulations are then used to build full-length structural models. [START_REF] Yang | I-TASSER server: new development for protein structure and function predictions[END_REF] -AlphaFold network predicts the 3D coordinates of all atoms for a given protein. It relies on artificial intelligent by learning from multiple core amino acid sequences/structures and aligns sequences of homologues as inputs. [START_REF] Jumper | Highly accurate protein structure prediction with AlphaFold[END_REF] The choice of component proteins to be fused is generally simple when the specific application is in mind. Fusion partners, in theory, should have been thoroughly

researched and reported to be physico-chemically compatible (e.g., optimum pH, temperature, ionic strength, and inhibitor effects). [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] The order in which fusion partners appear in the polypeptide chain is important, as the location of one domain can have an impact on the localization and functionality of another. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] As a result, unless the possible effect of the order is known, two sequence choices should be tested in the event of a two-component fusion. An end-to-end genetic fusion, in which their coding genes are fused together and produced as a single peptide chain in a suitable host organism, is the easiest method of combining them after they've been chosen. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF]

Construction of fusion proteins

Fusion proteins can be obtained by different strategies mimicking nature's strategy such as tandem fusion, domain insertion and post translational conjugation (Figure 8). [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Chapter 1: State of the art Figure 8: Common strategies for the construction of fusion proteins. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] -Tandem fusion Direct tandem fusion is easy and effective in some circumstances where the N-or Cterminal regions are unstructured and flexible. These regions can operate as a "bridge" to give enough space between protein domains for proper folding. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] This strategy fails, however, when either the free N-or C-terminus to be tethered is required for the function of the parent protein and/or is not flexible or long enough to avoid steric hindrance, which reduces the degrees of freedom in protein dynamics and can result in undesirable consequences such as protein misfolding, aggregation leading to inclusion body formation, low protein yield, and impaired bioactivity. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] As a result, linker peptides are commonly used to connect fusion partners to preserve their distinct structures and functions (Figure 8). [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Chapter 1: State of the art

-Domain insertion

The majority of multidomain proteins are made up of domains encoded in sequential sequences on a single polypeptide chain; however, there are exceptions, such as when one domain is enclosed within the other. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] In 2004, Aroul-Selvam et al. [START_REF] Aroul-Selvam | Domain Insertions in Protein Structures[END_REF] reported that insertions are found in 9% of multidomain proteins with known structures, particularly in the α/β classes of proteins. This shows that the polypeptide sequence's continuity isn't required for proper domain folding. As a result, a functional fusion protein can be achieved by introducing one domain into a host protein (Figure 8).

It's worth mentioning that domain insertion is more difficult to design than tandem fusion because it requires finding a "cut" site on the host protein to accept the insert, which, if chosen incorrectly, could affect the folding of both domains. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] As a rule of thumb, insertion spots should be surface loops or turns that are not directly involved in the catalytic sites, to avoid clashes between the host and the inserted domains. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] The insert domain is usually shorter than the host, with its exposed N-and C-termini ligated to the host protein's internal regions through two ligation sites. In most cases, a successful fusion design using this method involves two conditions: [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] 1) the insert domain's N-and C-termini must be close together, and 2) the parent domain must be able to withstand the discontinuity induced by domain insertion. To preserve their individual functions, the structure of both domains should be intact and mutually permitted after the insertion without significant disruption. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Another important step in this method is to choose suitable ligation sites. In this regard, the program SCHEMA can assist in the selection of proper boundaries for chimera building without affecting the three-dimensional structure's integrity. [START_REF] Voigt | Protein building blocks preserved by recombination[END_REF] SCHEMA scans the protein structure and finds the interactions between residues that may be broken in the resulting chimeric protein, allowing for the prediction of possible chimera misfolding. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Figure 9 shows an example of engineering a chimeric enzyme using SCHEMA tool to find the insertion sites.

Figure 9: The engineering of a chimeric enzyme comprising two cellulases, CelA and SSO1949 using the 'domain insertion' approach. The SCHEMA tool was used to find the insertion sites on CelA. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] -Post translational protein conjugation:

Recombinant proteins can be produced via an alternate approach (Figure 8): individual proteins can be biochemically joined after they have been individually expressed. This approach is a protein-level ligation in which protein expression and fusion are separated into two phases. Although the two genetic fusion mechanisms outlined above are the most common approaches for chimera construction, a given pair of protein domains may fail to express in a tolerable yield or lack correct folding and function, limiting the range of domains that can be joined. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] When working with recombinant proteins derived from various hosts, this problem becomes even more serious. Post-translational protein fusion, on the other hand, allows for modular pairing and assembly of component domains after their expression in optimum hosts, effectively bypassing the genetic fusion's diversity-limiting cloning processes. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Bifunctional reagents capable of covalently binding to reactive functional groups on proteins (e.g., -SH, -NH2, and -COOH), which are routinely employed for protein immobilization and stabilization, are typically used for intermolecular protein conjugation. Although direct chemical cross-linking can result in functional fusion proteins, it is not always site-specific and can lead to protein aggregation. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Because the genetic fusion strategy may result in undesirable domain interactions or low protein production, post-translational fusion can be used in multiple-component systems. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Many examples have been reported such as the fusion of bovine serum Chapter 1: State of the art albumin with a lipase, the fusion of two immunoglobulin-binding protein G domains and the conjugation of two Green Fluorescent Proteins (GFP). [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Aside from utilizing the reactive side chains of natural amino acids, the introduction of (CYP51) and Fe-S containing NADPH reductase. This chimeric enzyme was able to catalyze oxidative demethylation of lanosterol more efficiently than CYP51 and the reductase separately, culminating in the development of a self-contained P450

biocatalysis system (scheme 8). [START_REF] Yu | Synthetic fusion protein design and applications[END_REF][START_REF] Choi | Novel iron-sulfur containing NADPH-Reductase from Nocardia farcinica IFM10152 and fusion construction with CYP51 lanosterol demethylase[END_REF] Scheme 8: Reaction catalyzed by the fusion enzyme composed of CYP51 and Fe-S containing NADPH reductase. 44

Biosensing and bioactivity regulation:

The binding of specific molecules by some proteins induces conformational changes.

Researchers use such proteins as switches that can operate as biosensors or regulators upon conformational change. The phenomenon of allostery, in which conformational changes at a remote regulatory site can be carried through to the active site, influencing bioactivity in either a negative or a positive manner, is one technique for developing such switches. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] 1CFD) and bound to Ca 2+ (pdb: 1CLL). [START_REF] Meister | An Engineered Calmodulin-Based Allosteric Switch for Peptide Biosensing[END_REF] When compared to the inactive state, the activated state of the allosteric enzyme demonstrated a 120-fold increase in BLA catalytic activity. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] This engineered calmodulin-based allosteric switch fusion protein can be used as biosensor to detect toxins and other biomarkers. 45

Creating peptide libraries

Creating protein libraries on the surface of cells and bacteriophages is considered an important application of chimeric proteins. [START_REF] Bonde | Chimeric Genes[END_REF] Recently, R.M. Lu et al [START_REF] Liu | Synthetic chimeric nucleases function for efficient genome editing[END_REF] worked on developing several chimeras of nucleases. They have designed a Cas12a-type nuclease library by fusing different Cas12a-like nucleases and successfully screened functional chimeras and identified several dozen that showed basal editing. [START_REF] Liu | Synthetic chimeric nucleases function for efficient genome editing[END_REF] Then, they demonstrated the use of this strategy as a platform for rapid generation of nucleases. Some chimeras showed better specificity than all other parent-wild type nucleases. This demonstration opens-up the possibility of generating new nuclease sequences with implications across biotechnology. [START_REF] Liu | Synthetic chimeric nucleases function for efficient genome editing[END_REF] Chapter 1: State of the art

Bio-therapeutics applications

Therapeutic fusion proteins are categorized based on their incorporated protein domains such as: Fc (fragment crystallizable region) fusions, antibody-enzyme fusions, immunotoxins-cytokine fusions, and many other categories. [START_REF] Bonde | Chimeric Genes[END_REF] -Immunotechnology

Recently, chimeric proteins were shown to be promising tools for developing new antibodies that are used in a variety of analytical, therapeutics and clinical applications. [START_REF] Bonde | Chimeric Genes[END_REF][START_REF]Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges[END_REF] For example, fusions involving antibodies as one fusion partner, have made affinity-based analytical procedures such as enzyme-linked immunosorbent assay (ELISA) and Western blotting easier to design. [START_REF] Bonde | Chimeric Genes[END_REF] -Creating new drugs with targeted delivery to tumor cells Recombinant immunotoxins are developed via the fusion of a toxin with an antibody or its fragment that has an affinity for the target tumor cells. [START_REF] Bonde | Chimeric Genes[END_REF] i.

In 2003, Halin et al. [START_REF] Halin | Synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin 12 and to tumor necrosis factor alpha[END_REF] fused two cytokines: Tumor necrosis factor (TNF) and interleukin-12 (IL-12), with an scFv human antibody (Figure 11). This recombinant protein showed strong tumor targeting properties, which were attributed to the cytokine activity and antibody binding capacity. comprising the IL-12, TNF and scFv human antibody. [START_REF] Halin | Synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin 12 and to tumor necrosis factor alpha[END_REF] ii.

In 2004, a highly stable conjugate with potent tumor activity was created via the fusion of BLA enzyme with the heavy chain of a single-domain antibody. This recombinant enzyme has shown a good biodistribution profile in vivo and a cancer therapy. It can be used to cure tumor xenografts. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF][START_REF] Cortez-Retamozo | Efficient Cancer Therapy with a Nanobody-Based Conjugate[END_REF] Chapter 1: State of the art -Humanized antibodies Many antibodies were and still are developed based in mice. These antibodies evoke an immune response when administered to humans. Chimeras wherein human antibody constant domains replaced the corresponding mouse moieties eliminated most of the immunogenic part of the antibodies without modifying specificity. [START_REF] Bonde | Chimeric Genes[END_REF] -Some examples of fusion therapeutic's proteins:

There are currently a number of fusion protein-based therapeutics on the market. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Some examples are cited below:

i.

Etanercept: a drug formed by a tumor necrosis factor receptor (TNFR) linked with an immunoglobulin G1 (IgG1) Fc segment. [START_REF] Bonde | Chimeric Genes[END_REF][START_REF] Yu | Synthetic fusion protein design and applications[END_REF] It is indicated to treat autoimmune diseases such as psoriasis, rheumatoid, polyarthritis… ii.

Ontak: developed via the fusion of the human IL-2 and the catalytic and transmembrane domains of the toxin of diphteria. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] It is indicated to treat recurrent cutaneous T-cell lymphoma.

iii.

Amevive: results from the fusion of the Fc domain of IgG1 and the human LFA-3. It is used to treat symptoms of psoriasis. 28

CUPIN PROTEINS

As we mentioned before, to prepare the chimeric enzyme, we chose to rely on the cupin superfamily of proteins. We selected two related proteins from the bicupin family: PMI and PLP. For this, we will describe below the cupin superfamily of proteins.

The cupin superfamily is a diverse protein superfamily whose name is derived from that of its conserved -barrel domain, as cupa is the Latin term for a small barrel. This protein superfamily is amongst the most versatile of all protein folds in activity, structure and distribution. It comprises a wide variety of enzymes besides nonenzymatic proteins. [START_REF] Dunwell | Cupins: the most functionally diverse protein superfamily?[END_REF] The first cupin to be studied was discovered to be a germination marker in higher plants' seeds by Thompson and Lane 50 in 1980 and was thus given the name "Germin".

Initial studies showed that it had a homopentameric structure [START_REF] Faye | Common antigenic determinants in the glycoproteins of plants, molluscs and insects[END_REF][START_REF] Jaikaran | Covalently bonded and adventitious glycans in germin[END_REF] and that it possessed an oxalate oxidase (ox-ox) activity.

Another protein from the cupin family i.e., spherulin, involved in the response of the [START_REF] Dunwell | A New Superfamily of Functionally Diverse Proteins that Include Germins and Plant Storage Proteins[END_REF] Finally, Woo and coworkers [START_REF] Dunwell | Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities[END_REF] published in 2000 a crystal structure at a resolution of 1.6

Å that proved that germin was a homohexameric cupin and shed light on the catalytic mechanism of its ox-ox activity.

The characteristic cupin domain comprises two conserved motifs 1 and 2, each corresponding to two β-strands, separated by a less conserved inter-motif region composed of another two β-strands with an intervening variable loop (Figure 12). 11 Initially, the conserved characteristic sequence of Motif 1 was identified as G(X)5HXH(X)3,4E(X)6G and that for motif 2 as G(X)5PXG(X)2H(X)3N. [START_REF] Dunwell | Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities[END_REF] However, in 2004, another study [START_REF] Dunwell | Cupins: the most functionally diverse protein superfamily?[END_REF] showed that the primary sequence of the two motifs was much less conserved. For example, one of two His residues in Motif 1 that are supposedly conserved can be substituted by Gln or Asp/Glu in some isomerases and dioxygenases.

The characteristic features of these proteins with this fold include resistance to Chapter 1: State of the art proteases as well as thermal stability. These characteristics are attributed to their high degree of subunit contacts, hydrophobic interactions, and short loops. [START_REF] Uberto | Protein Similarity Networks Reveal Relationships among Sequence, Structure, and Function within the Cupin Superfamily[END_REF] According Analysis of the structure-activity relationships among the cupin family had allowed to identify that they were involved in some 18 different functional classes that range from single domain bacterial enzymes (isomerases and epimerases) through two- In 2004, proteins from the cupin family have been classified according to the number of cupin domains they contained, so 3 subgroups of cupins: Monocupins, bicupins and multicupins could be distinguished. 11 a) Monocupins:

These are proteins that comprise a single cupin domain, either as the center of a simple protein or as one of several domains in a complex protein. Monocupins include some microbial transcription factors (for example AraC) and enzymes such as Dioxygenases 2-oxoglutarate iron dependant dioxygenases such as ACCO (Figure 13), Acireductone dioxygenases, Auxin binding protein… Although the majority of enzymatic cupins contain iron as a metal cation in their active site, other members contain either cations of copper, zinc, cobalt, nickel or manganese as cofactors allowing the catalysis of various kinds of chemical reactions to occur within the conserved tertiary structure. 11

CONCLUSION

Biocatalysis using natural enzymes is mostly limited to the natural substrate of the employed enzyme and to biotic reactions. Starting from novel substrates, artificial enzymes catalyze chemist-invented reactions leading to industrially relevant products under ecofriendly conditions. [START_REF] Matsuo | Artificial enzymes with protein scaffolds: Structural design and modification[END_REF] Metal complexes are the most efficient man-made catalysts that are routinely used to form the catalytic sites of artificial metalloenzymes. The latter combine both the advantages of natural metalloenzymes such as stereoselective catalysis in aqueous solution, under mild conditions and the advantages of chemically synthesized metal complexes catalyst such as chemist-invented catalysis and compatibility with artificial substrates. [START_REF] Ghattas | Receptor-Based Artificial Metalloenzymes on Living Human Cells[END_REF] During this thesis, several artificial metalloproteins have been constructed by different strategies. This chapter presented an overview of artificial enzymes by discussing their advantages and their construction strategies. In particular, Chimeric enzymes are introduced as they are further advantageous in atom economy.

In this project, we followed a new strategy to develop artificial metalloenzymes: We aim to prepare an artificial chimeric enzyme that possesses multiple active sites by the fusion of catalytic domains of 2 related enzymes. This chimeric enzyme would be used to catalyze reactions in cascade under ecofriendly conditions.

To prepare the chimeric enzyme, we chose to rely on the cupin superfamily of proteins.

Cupins are proteins characterized by a structure formed of barrels of four β sheets containing the active site and are surrounded by alpha helixes. [START_REF] Dunwell | Cupins: the most functionally diverse protein superfamily?[END_REF] Pirin Like protein (PLP)

and Mannose-6-Phosphate Isomerase (PMI) are similar bicupins characterized by a structure formed of two β barrel cupin domains but each of the two enzymes possess one active site in only one of the cupin domains. [START_REF] Ahmad | Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-D -arabinonhydrazide[END_REF][START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] Therefore, we plan to design a functional chimeric enzyme by combining the catalytic domains of PMI and PLP, which should avoid denaturation and lead to a correct folding.

CHAPTER 2: PHOSPHOMANNOSE ISOMERASE

INTRODUCTION:

Prior to the preparation of the chimeric PMI-PLP metalloproteins we proceeded to express and purify each of the two starting enzymes i.e. PMI and PLP and to test their activities before and after exchanging the metal cations of their respective active site.

We plan to compare these activities with those of the chimeric PMI-PLP metalloproteins.

In this chapter, after a short presentation of the structures and properties of the various variants of PMIs, we will then describe the preparation and the characterization of a novel metalloprotein based on PMI from Candida albicans by substituting the native Zn 2+ of its active site by a Cu 2+ .

The construction of the expression vector encoding for PMI and His6 tag flanking TEV protease cleavage site (TEVc/site) will first be explained. The cloning and the overexpression of the His6-TEVc/site-PMI in E. coli. will be presented. After exposing the purification and characterization of the obtained protein, its PMI activity will be discussed. Subsequently, PMI transformation into an artificial metalloprotein by exchanging the native Zn 2+ by Cu 2+ will be described. The preparation and characterization of the artificial metalloprotein by various spectroscopic techniques will be detailed and finally, an activity study will be presented.

STATE OF THE ART

PMI's are metal-dependent aldose-ketose isomerases catalyzing the interconversion of D-mannose-6-phosphate (M6P) and D-fructose-6-phosphate (F6P) in eukaryotic and prokaryotic organisms. [START_REF] Ahmad | Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-D -arabinonhydrazide[END_REF] In 1968, PMI was suggested, for the first time, to be a metalloenzyme on the basis of studies that showed that chelating agents inhibited its activity and that this activity was regenerated by addition of metal cations, such as Co 2+ . [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] It was also found that this metalloenzyme was a bicupin containing one atom of zinc per enzyme monomer ( MW = 45 kDa). [START_REF] Jensen | Domain organisation in phosphomannose isomerases (types I and II)[END_REF] This metal was located at the active center of the enzyme and it appeared to be essential for the catalysis since a substrate can protect the enzyme from inactivation by metal chelators. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF][START_REF] Jensen | Domain organisation in phosphomannose isomerases (types I and II)[END_REF][START_REF] Cleasby | The x-ray crystal structure of phosphomannose isomerase from Candida albicans at 1.7 angstrom resolution[END_REF] PMI was thus the first isomerase for which a metal was established as an integral part of the enzyme structure. [START_REF] Jensen | Domain organisation in phosphomannose isomerases (types I and II)[END_REF][START_REF] Cleasby | The x-ray crystal structure of phosphomannose isomerase from Candida albicans at 1.7 angstrom resolution[END_REF] Different studies have shown that PMI is required for the survival, virulence, and/or pathogenicity of several bacteria and protozoan parasites [START_REF] Shinabarger | Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa[END_REF][START_REF] Patterson | Mannose metabolism is required for mycobacterial growth[END_REF] , as well as for the yeast cell wall integrity in Saccharomyces cerevisiae [START_REF] Payton | A novel Saccharomyces cerevisiae secretory mutant possesses a thermolabile phosphomannose isomerase[END_REF] , Candida albicans [START_REF] Smith | Cloning and heterologous expression of theCandida albicans genePMI 1 encoding phosphomannose isomerase[END_REF] , Cryptococcus neoformans [START_REF] Wills | Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity: Isolation of the MAN1 gene in Cryptococcus neoformans[END_REF] , and Aspergillus nidulans. [START_REF] Smith | Hyphal tip extension in Aspergillus nidulans requires the manA gene, which encodes phosphomannose isomerase[END_REF] In addition, PMI was found an essential enzyme for E.coli: [START_REF] Schmidt | The Rhizobium meliloti pmi gene encodes a new type of phosphomannose isomerase[END_REF] Mutants lacking PMI could not multiply on a medium where mannose was the only source of carbon and could not synthesize their capsular completely neither. [START_REF] Schmidt | The Rhizobium meliloti pmi gene encodes a new type of phosphomannose isomerase[END_REF] As a result, PMI is being explored as a possible target against fungal infections that cause significant disease or death, particularly in immunocompromised people. 59,67

Different types of PMI: classification of Proudfoot

Based on their amino acid sequence identity, PMIs have been classified into three unrelated families as proposed by Proudfoot et al. [START_REF] Proudfoot | cDNA cloning and heterologous expression of human phosphomannose isomerase[END_REF] The organisms that produce the most common Type I, Type II and Type III PMI enzyme families are summarized in Table 3. [START_REF] Jensen | Domain organisation in phosphomannose isomerases (types I and II)[END_REF][START_REF] Proudfoot | cDNA cloning and heterologous expression of human phosphomannose isomerase[END_REF] This group contains PMIs of identified eukaryotic organisms including human PMI, PMIs of several yeasts as well as PMIs of most bacteria and parasites. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] Type I PMIs are monofunctional enzymes that catalyze only the reversible isomerization of M6P into F6P. They are monomeric enzymes, with a molecular weight of about 45 kDa that include a zinc cation (Zn 2+ ) within their active site. Their optimal pH varies between 7 and 8.5. [START_REF] Proudfoot | Purification and characterization of fungal and mammalian phosphomannose isomerases[END_REF] Type I PMI are homologous proteins with greater than 30 % sequence identity. The ligands of the zinc cation within the active site are conserved from one enzyme to another. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF][START_REF] Proudfoot | Purification and characterization of fungal and mammalian phosphomannose isomerases[END_REF] The replacement of Zn 2+ with other metal cations such as Ni 2+ , Cd 2+ , Mn 2+ , Cu 2+ or Fe 2+ causes a loss of their PMI activity. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] However, an increase in the isomerase activity was noted when Zn 2+ was replaced with Co 2+ . [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] Thus, these enzymes poorly tolerate changes in their metal cofactor. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF]

Type II PMIs:

Type II PMIs are classified as bifunctional enzymes with a molecular weight of 55 kDa. They are found exclusively in bacteria. They are involved at two distinct levels of the metabolism of mannose: in addition to their isomerase activity, these enzymes also possess a guanosine diphosphate (GDP)-D-mannose pyrophosphorylase (GMP) activity, the last phase of the production of GDP-mannose (GDP-D-mannose). [START_REF] Proudfoot | cDNA cloning and heterologous expression of human phosphomannose isomerase[END_REF] Each activity occurs in a separate domain. [START_REF] Jensen | Domain organisation in phosphomannose isomerases (types I and II)[END_REF] GDP-D-mannose is a precursor of mannoconjugated substrates (GDP-L-fucose, GDP-colitose, GDP-perosamine, and GDP-Drhamnose) which are important for cell survival of both eukaryotes and prokaryotes and for the pathogenicity of most microorganisms. [START_REF] Jensen | Domain organisation in phosphomannose isomerases (types I and II)[END_REF] Type II PMIs require the presence of a metal in their active site to be functional. Unlike type I PMIs, these enzymes tolerate variation in the metal cofactor. They are active not only with zinc, but also with a wide range of divalent cations such as Co 2+ , Mg 2+ , Ni 2+ , Mn 2+ and Ca 2+ . [START_REF] Shinabarger | Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa[END_REF] These enzymes present a low sequence similarity with those of type I PMIs that is limited to a small number of amino acids that belong to their PMI domain. [START_REF] Patel | 9.12 Industrial Applications of Biocatalytic Hydrolysis (Esters, Amides, Epoxides, Nitriles) and Biocatalytic Dynamic Kinetic Resolution[END_REF] The conservation of these residues between type I and type II PMIs thus suggest a common catalytic process for their isomerase activity. [START_REF] Jensen | Domain organisation in phosphomannose isomerases (types I and II)[END_REF] Some bacteria, such as E. coli and S. typhimurium, possess the 2 types of PMI. The reason for this remains unclear. Proudfoot et al. [START_REF] Proudfoot | cDNA cloning and heterologous expression of human phosphomannose isomerase[END_REF] proposed a hypothesis assigning different roles to the two types of enzymes: PMIs type I could preferably be involved in the metabolism of D-mannose, whereas PMI type II could be involved in the synthesis of polysaccharide capsules.

Type III PMIs:

This type of enzymes is limited to the Gram-negative bacterium Rhizobium meliloti and presents a low sequence similarity with those of type I and type II PMIs. [START_REF] Schmidt | The Rhizobium meliloti pmi gene encodes a new type of phosphomannose isomerase[END_REF][START_REF] Proudfoot | cDNA cloning and heterologous expression of human phosphomannose isomerase[END_REF] In 1992, Schmidt et al. isolated and established the complete nucleotide sequence of the PMI gene of R. meliloti. This nucleotide segment showed two open reading frames (ORFs) that code for 18-and 43-kDa polypeptides. According to gene disruption studies, the ORF2 encoded for PMI. Hence, there was no substantial resemblance between the deduced amino acid sequence and the equivalent sequences of the Pseudomonas aeruginosa and Escherichia coli PMIs, indicating that the isolated gene produces a novel type of PMI. 66

A novel type: Type IV PMIs:

These enzymes have been recently described in the literature. [START_REF] Islam | Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from D ehalococcoides mccartyi: Functional characterization of D. mccartyi proteins[END_REF][START_REF] Roux | The reaction mechanism of type I phosphomannose isomerases: New information from inhibition and polarizable molecular mechanics studies: Type I Phosphomannose Isomerases Mechanism[END_REF] They present proteins with dual specificity: PMI and Phosphoglucose isomerase (PGI). [START_REF] Roux | The reaction mechanism of type I phosphomannose isomerases: New information from inhibition and polarizable molecular mechanics studies: Type I Phosphomannose Isomerases Mechanism[END_REF] In general, PGI has a crucial role in sugar metabolism and it catalyzes the interconversion of G6P into F6P. [START_REF] Islam | Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from D ehalococcoides mccartyi: Functional characterization of D. mccartyi proteins[END_REF] These enzymes belong to the PGI superfamily because their folding is similar to the inner core of PGIs from eucaryotes and bacteria and they possess a PMI activity. [START_REF] Swan | A Novel Phosphoglucose Isomerase (PGI)/Phosphomannose Isomerase from the Crenarchaeon Pyrobaculum aerophilum Is a Member of the PGI Superfamily[END_REF] They include DmPMI (Dehalococcoides mccartyi PMI) which was proposed to be a hypothetical protein and a bifunctional PMI/PGI as well as the PMIs of the actinobacteria, firmicutes and bacteroides families. [START_REF] Islam | Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from D ehalococcoides mccartyi: Functional characterization of D. mccartyi proteins[END_REF] They share a low sequence similarity with those of other types of PMI and their activity is lower. [START_REF] Islam | Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from D ehalococcoides mccartyi: Functional characterization of D. mccartyi proteins[END_REF] This lower in activity was suggested to be correlated to the fact that the substrate could be a different sugar than M6P. [START_REF] Islam | Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from D ehalococcoides mccartyi: Functional characterization of D. mccartyi proteins[END_REF] 2.2. PMI is an enzyme widely used in nature as it is involved in the metabolism of mannose allowing the synthesis of mannosylated structures and thus glycoconjugates. [START_REF] Ahmad | Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-D -arabinonhydrazide[END_REF] However, it is less widespread in plants and the gene coding for the PMI of Escherichia coli was recently used as a marker gene for the selection of genetically modified plants.

Only plants that are transformed by the PMI gene are able to grow on media containing mannose as a single source of carbon. [START_REF] Duan | An efficient and high-throughput protocol for Agrobacteriummediated transformation based on phosphomannose isomerase positive selection in Japonica rice (Oryza sativa L.)[END_REF]

PMI: Characterization as a Zinc metalloenzyme

In 1958, Noltmann and Bruns [START_REF] Bruns | Phosphomannoisomerase, an SH-Dependent Metal-Enzyme Complex[END_REF] discovered that the activity of the PMI from pig erythrocytes was inhibited by chelating agents such as versene, dipyridyl and ophenanthroline [START_REF] Bruns | Phosphomannoisomerase, an SH-Dependent Metal-Enzyme Complex[END_REF] and that this activity could be regenerated after addition of divalent metal cations such as Ca 2+ , Zn 2+ , Co 2+ and Fe 2+ . This led them to suggest that PMI is a metal-enzyme complex. [START_REF] Bruns | Phosphomannoisomerase, an SH-Dependent Metal-Enzyme Complex[END_REF] In 1968, this observation was later confirmed by Gracy and Noltmann who purified for the first time the PMI from Saccharomyces cerevisiae. [START_REF] Gracy | AND DETERMINATION OF SOME PHYSICAL PROPERTIES[END_REF] The inhibition by chelating agents such as EDTA (Scheme 10), and o-phenanthroline was pH-dependent, occurring faster and easily at pH 6 than at pH 8. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] The chelating agent was treated with a stoichiometric quantity of metal which results in the coordination of the agent's binding sites, reducing its ability to interact with the enzyme's metal. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] Hence, the inhibition could be prevented by blocking the coordination sites of the metal-binding agents. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] The regeneration was instantaneous and was dependent on the metal species as well as on its concentration: Metals binding tightly to EDTA such as Zn 2+ , Co 2+ and Fe 2+ were the most powerful metals to reverse the inhibition of EDTA; although, metals which bind much lower to EDTA such as Ca 2+ and Mg 2+ were not capable to reverse the inhibition of EDTA. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] In addition, Bruns, Noltmann and Willemsen have shown that zinc was able to reverse completely the inhibition of the erythrocyte PMI by EDTA. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] Scheme 10: Reaction of EDTA with PMI.

Then, Gracy and Noltmann [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] showed via metal analyses performed by atomic absorption spectrophotometry that the PMI of yeast was a monomeric enzyme having a molecular weight of 45 KDa and containing a Zn 2+ cation per protein. The authors reported that this divalent cation played an important role in the catalysis of the reversible isomerization of M6P into F6P. Indeed, the enzyme was protected against chelating agent's inhibition by the substrates, which confirmed first, that the metal was indeed in the active site of the enzyme and that it bound the substrate, and second, that this metal was essential for the catalysis. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] Chapter 2: Phosphomannose Isomerase PMI was the first isomerase for which a metal was established as an integral part of the enzyme. [START_REF] Gracy | Studies On Phospomannose Isomerase: A Mechanism For Catalysis And For The Role Of Zinc In The Enzymatic And The Nonenzymatic Isomerization[END_REF] Its zinc cation maintains its structural conformity and confers a catalytic role. [START_REF] Ahmad | Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-D -arabinonhydrazide[END_REF]

PMI from Candida albicans

PMI from Candida albicans is a type 1 PMI. It is a bicupin that contains two domains, each composed of a barrel of β-sheets, which are connected by a single small interdomain composed of α-helixes (Figure 16). It also contains one atom of zinc in the active site, located in the β-sheet barrel domain at the C-terminal side of the protein (Figure 16). 11 F6P was located in the binding site in a position that suggested its coordination to Zn 2+ . However, the electron density around the F6P was unclear, which was justified by the authors by the co-existence of the cyclic fructofuranose form of ß-F6P and its partially open form. [START_REF] Cleasby | The x-ray crystal structure of phosphomannose isomerase from Candida albicans at 1.7 angstrom resolution[END_REF] Nevertheless, this coordination proves that the zinc has not only a structural role but also a catalytic role in the reaction process. [START_REF] Ahmad | Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-D -arabinonhydrazide[END_REF] In the hosting laboratory, phospho-D-arabinohydrazide (5PAHz) was designed as an analogue of the enediolate high-energy intermediate in the mechanism of the M6P/F6P isomerization catalyzed by PMI (Scheme 11). This compound caused a strong inhibition of PMI (Ki = 1.7 μM). A His6 tagged variant of PMI was also expressed, purified, and crystallized in the presence of 5PAHz. The structure of PMI complexed with 5PAHz at 1.85 Å resolution was obtained (Figure 19). [START_REF] Ahmad | Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-D -arabinonhydrazide[END_REF] Figure 19: Crystal structure of His6-PMI coordinated to the inhibitor 5PAHz. Chapter 2: Phosphomannose Isomerase This structure suggests that Glu294 was the catalytic base that transferred a proton between the C1 and C2 carbon atoms of the substrate (Scheme 11). It also showed the bidentate coordination of the inhibitor on Zn 2+ which explained the stereoselectivity of the reaction (Figure 19). [START_REF] Ahmad | Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-D -arabinonhydrazide[END_REF] 2.2.4. Depletion of PMI and its consequences PMI constitutes a potential therapeutic target. The effects of the lack of PMI have been studied in most fungi and yeasts. These studies have shown that fungal microorganisms lacking PMI were unable to secrete the glycoproteins constituting their cell wall. However, supplementing the media with D-mannose allowed in some cases to compensate for PMI deficiency. [START_REF] Fang | Characterization of the Aspergillus fumigatus phosphomannose isomerase Pmi1 and its impact on cell wall synthesis and morphogenesis[END_REF] Nevertheless, mannose supplementation does not make it possible either to overcome PMI failure in Aspergillus nidulans, or to restore the morphological defects caused by the absence of this enzyme in C.

neoformans. [START_REF] Wills | Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity: Isolation of the MAN1 gene in Cryptococcus neoformans[END_REF] These studies show the importance of PMI for the growth and survival of these microorganisms, it then appears as a potential therapeutic target for conceiving new antifungals. [START_REF] Wills | Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity: Isolation of the MAN1 gene in Cryptococcus neoformans[END_REF] A study by the team of Wills et al. supports this hypothesis: the absence of PMI in C. neoformans mutants not only causes an arrest of growth "in vitro", but also a loss of virulence of the pathogens "in vivo", showing for the first time the validity of PMI as a potential antifungal target. [START_REF] Wills | Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity: Isolation of the MAN1 gene in Cryptococcus neoformans[END_REF] As for human PMI, this latter has not been widely studied; it has been shown that patients suffering from leukemia present reduction in PMI activity. [START_REF] Wills | Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity: Isolation of the MAN1 gene in Cryptococcus neoformans[END_REF]

RESULTS AND DISCUSSION:

Redesign the expression vector for the PMI

The pRSET-C vector comprising an insert encoding for the His6-tagged PMI (His6-PMI) was available in host laboratory. This plasmid comprises a His6 tag which should be cleaved before the metal substitution because it's known to be an excellent ligand for metal cations. For this, we planned to insert a specific TEVc/site flanked by the sequence of the enzyme and that of the His6 tag.

The initial plasmid pRSET-C was first digested by the restriction enzyme NheI. The result of this reaction was analyzed by agarose gel electrophoresis and ethidium bromide (ETBr) staining (Figure 20). A single band around 8 Kb was visible after digestion by Nhe1 (lanes 1 and 2) which confirmed that the digestion of the plasmid was successful.

For the undigested plasmid, three different bands can be seen (lane 3) which can be assigned to the three different structures of the plasmid DNA: nicked-relaxed, supercoiled and circular single-stranded.

The digested plasmid was extracted from the gel and then used to insert the DNA encoding for TEVc/site (ENLYQFS). The DNA insert encoding for the TEVc/site was a synthetic double stranded DNA that was complementary to the remaining palindromic fragments of the Nhe1 restriction sites in the linearized plasmid (Figure 21). In the presence of ligase, the synthetic insert could ligate in the vector in two directions as both ends were complimentary to the palindromic remaining fragments of the Nhe1 restriction site: either in the direction coding for TEVc/site or in the direction opposite to that coding for TEVc/site resulting in the amino acid sequence ASHALKIQVF. It is also noteworthy that, because of these palindromic fragments at the ends of the vector, the addition of ligase could also yield the starting circular plasmid thus without the incorporation of the insert. A mixture was formed by adding 10 equiv. of double stranded synthetic DNA insert encoding for TEV protease to 1 equiv. of the purified digested plasmid. Bacteriophage T4 DNA ligase and ATP were added to the mixture. The excess of insert was aimed at limiting the self-ligation of the vector without incorporation of insert.

The ligation mixture was then used to transform DH5alfa competent bacteria. The growth of bacteria following transformation in antibiotic containing medium allows the selection of recombinant cells if the newly acquired DNA conveys an antibiotic Chapter 2: Phosphomannose Isomerase resistance. In the present case, among the selected bacteria about 50 colonies were further screened by PCR for the correct insertion of the TEV protease DNA.

Colony screening using PCR is a convenient high-throughput method that can be used for determining the presence or absence of an inserted DNA in plasmid constructs and can also help to determine the orientation of the insertion. PCR amplification of a plasmid fragment using an insert specific primer paired with a vector specific primer provided amplicons of a specific size (700 bp) only if the insert is indeed present in the plasmid and in the correct direction. This was achieved using primer mix 1: CCTGTATTTTCAGGGCATGG and TTCAGGCCAACATGATTCAG (Figure 22). The success of the PCR is dependent on the presence of the expected insert and on the reaction conditions. To optimize PCR conditions and to avoid negative results due the experimental conditions, we relied on a set of primers that targeted a DNA sequence that was surely present in the plasmid. All selected colonies acquired ampicillin resistance by incorporating the transformation plasmid. Accordingly, the reaction conditions were optimized using primer mix 2 (positive control), that targeted the sequence of the gene encoding for ampicillin resistance to provide amplicons of about 700 bp and showed physical properties similar to those of primer 1 (Tm = 54.1-55 °C, Annealing T = 48-49 °C).

PCRs were performed and the samples were analyzed by electrophoresis/Syber safe staining. Figure 23 shows that using primer mix 2, a fragment of amplified DNA of about 700 bp was formed, proving that, as expected, all selected colonies contained the sequence of the gene encoding for ampicillin resistance. Using the same conditions with primer mix 1, a fragment of amplified DNA of 700 bp was detected only for colonies of lanes 7 and 12 (Figure 24). The plasmid containing the insert encoding for the TEVc/site was thus only incorporated in the desired direction in the bacteria of these two colonies. In order to confirm these results, and to examine the junctions between the plasmid and the inserted DNA, colonies that provided lanes 7 and 12 were grown and their plasmid DNA extracted and sequenced. Indeed, they show the expected sequence.

Expression and purification of the His6-PMI:

E. coli BL21(DE3) strain was used for expressing the protein. B strains are deficient in lon protease (cytoplasm) and OmpT protease (outer membrane) therefore they are preferred for the expression of recombinant proteins. The strain used contained the λDE3 lysogen that carries the gene for T7 RNA polymerase under control of the lacUV5

promoter. IPTG addition is thus required to maximally induce expression of the T7 RNA polymerase in order to express recombinant genes cloned downstream of a T7

promoter. Accordingly, the PMI gene was located downstream of the T7 promoter.

Purified and sequenced plasmid DNA containing the insert sequence was used to transform BL21(DE3) that were then selected on agarose/ampicillin.

A pre-inoculum from a single colony was grown in a starter culture LB-ampicillin. TB supplemented with ampicillin was inoculated from the preinocculum at 1/100 and grown at 37 °C until the OD at 600 nm reached a value between 0,6 and 0,8. 1 mM IPTG was then added and incubation was continued at 20 °C for 18 h. Bacterial cells were pelleted and kept at -20 °C. Analysis by SDS-PAGE, followed by Coomassie blue staining, showed a band of high intensity at a MW of about 50 kDa in all the lanes corresponding to the bacterial pellets, which was characteristic of the expression of His6-PMI. By contrast, no intense protein band was observed in the lanes corresponding to the supernatant fractions (Figure 25). The bacterial pellets were resuspended in the presence of benzonase and lysozyme. The mixture was sonicated then centrifuged to eliminate all insoluble fractions. The clear solution was passed through Ni-NTA agarose column. The various fractions eluted from the column were analyzed by SDS-PAGE followed by Coomassie blue staining. As shown in Figure 26, a band corresponding to a protein with a molecular weight of about 50 kDa was observed for fractions 6 to 12, which suggested that those fractions contained the purified His6-PMI. To improve the expression of the His6 -PMI, E. coli BL21(DE3) pLysS strain was used for expressing the protein. This strain allows high efficiency protein expression, express T7 RNA polymerase which can improve the expression and encode T7 lysozyme that suppresses basal expression of toxic target proteins prior to induction. The same procedure was followed as above to express and purify the protein. Figure 27 confirms that this strain is much better for the His6-PMI's expression. A band with high intensity corresponding to a protein with a molecular weight of about 50 kDa was observed for fractions 3 and 4, which suggested that those fractions contained the purified His6-PMI.

Purification of His6 tag free PMI:

The purified His6-PMI protein fractions were incubated with His6-AcTEV. Following digestion, His6-AcTEV was easily removed from the reaction medium by purification on a Ni-NTA affinity column.

Indeed, SDS-PAGE analysis of the fractions eluted from the Ni-NTA affinity column, followed by coomassie blue staining revealed a characteristic band for the flow through at a molecular weight of about 50 KDa (Figure 28 left, lane 3), similar to those of the starting His6-PMI protein (Figure 28 left, lane 1) and of the reaction mixture (His6-PMI + His6-AcTEV) (Figure 28 left, lane 2), while no band was found for the fraction eluted with 300 mM imidazole (Figure 28 left, lane 4).

The SDS-PAGE analysis was also followed by a Western blot using anti His6-tag antibody (Figure 28). The comparison with the coomassie blue staining experiment confirms that the starting protein was indeed His6 tagged (Figure 28 right, lane 1) while after incubation with AcTEV the His6-PMI had lost its His6 tag (Figure 28 right, lane 2). Similarly, no band was observed in the lane 3 of the western blot corresponding to the flow through of the Ni-NTA column, which suggested that it indeed contained the tag free PMI. However, it appears that the fraction eluted with imidazole still contained a low amount of His6-PMI that had not been digested (Figure 28 right, lane 4). The enzymatic activity of PMI was assessed via the measurement of the rate of isomerization of M6P into F6P at 25°C using a method described by Gracy and Noltmann. [START_REF] Gracy | AND DETERMINATION OF SOME PHYSICAL PROPERTIES[END_REF] This method is based on three successive reactions: (1) the isomerization of M6P into F6P catalyzed by PMI followed by (2) the isomerization of F6P into G6P

(glucose-6-phosphate) catalyzed by PGI (PhosphoGlucose Isomerase) and, finally, by

(3) the oxidation of G6P to 6PGL (6-phosphogluconolactone) by G6PDH (Glucose-6phosphate dehydrogenase) (Scheme 12). The later reaction requires NADP + that gets reduced into NADPH which can be detected by following its characteristic absorption at 340 nm (molar extinction coefficient = 6220 M -1 .cm -1 ). Both auxiliary enzymes (PGI and G6PDH) are added in large excess and the activity is measured using both pure tag free PMI and His6-PMI.

Zinc (II) removal:

It was previously reported by Gracy and Noltmann that up to 85 % of the PMI's activity could be lost by incubation with 10 000 equiv. of EDTA at 30°C for 8 h. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] Metal concentration measurements also indicated a loss of Zn 2+ upon incubation with EDTA. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] This confirms that the removal of the metal cofactor, Zn 2+ from the active site of PMI leads to a loss of the activity.

The effect of the addition of increasing amounts of EDTA (0-10 6 equiv.) on PMI at 4°C in 25 mM MOPS was thus investigated. A significant decrease of PMI activity was then observed upon incubation with increasing amounts of EDTA, which could be corelated to the amount of Zn 2+ removed. [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] Indeed, upon addition of 10 4 equiv. of EDTA and after dialysis for 2 days, the enzyme loss 80 % of its activity whereas the addition of 10 6 equivalent of EDTA to PMI was required to achieve 98% removal of Zn 2+ (Figure 30). Chapter 2: Phosphomannose Isomerase

Incorporation of Cu(II) into zinc free PMI

The Incorporation of Cu(II) into zinc free His6 tag free PMI was followed by fluorescence spectroscopy that is a very sensitive, rapid, and convenient method to study environmental changes in a protein, in particular those caused by the interaction of a protein with a ligand or a metal cation.

Upon addition of increasing amounts of copper nitrate (0.2-55 equiv.) in 50 mM MOPS, pH=7.4 to the zinc free PMI, 4.5 µM in 50 mM MOPS, pH = 7.4, the intensity of the protein intrinsic fluorescence signal increased (Figure 31). The fluorescence intensity value at 340 nm was then plotted against the concentration of added copper(II) nitrate, and the resulting curve plot (Figure 32) was fitted by using the following equation, which allowed the determination of the KD (dissociation constant) value for the PMI-Cu(II) complex as well as the Cu/PMI stoichiometry:

Where: F is the intensity of the fluorescence signal at 340 nm, [P]0 and [L]0 are respectively total protein and total copper(II) concentrations, n is the stoichiometry (number of ligand-binding sites per protein); Fmax = F0-Fmax; (F0 and Fmax are the Chapter 2: Phosphomannose Isomerase intensities resp. in absence of copper and when the protein is saturated with copper) represents the asymptotic value for F. Fmax, KD, and n were calculated from the fitting equation. A good fit was achieved with n = 1 and and KD = 3 µM, indicating that one Cu 2+ was bound tightly to one molecule of PMI to form a new Cu(II)-PMI protein the activity of which remain to be assayed. The new copper-PMI artificial metalloprotein was characterized by EPR spectroscopy.

For this, the X-band EPR spectra of frozen solutions of zinc free PMI and both Cu(II)His6-PMI and Cu(II)-PMI were recorded at 10 K. Zinc free PMI showed no EPR signal.

Cu(II)-PMI (Figure 33 B) showed an anisotropic EPR signal with an axial symmetry that confirms the presence of a single Cu 2+ binding site, that could be fitted with g// = 2.06, g⊥= 2.29 values, hyperfine coupling constants of A// = 16.7 mT.

In contrast, Cu(II)His6-PMI (Figure 33 A) showed an isotropic EPR signal of at least two Cu 2+ species, as seen in the g// part of the signal, and a total signal intensity equivalent to that of Cu-PMI. This suggests that the Cu 2+ is binding both His6 tag and the active center.

TYCHO:

TYCHO measurements were done to verify the structural integrity and to determine the inflection temperature of the PMI. In Figure 34, TYCHO showed that the natural PMI is well folded and its thermal denaturation can occur at 60°C. Upon substitution of Zn 2+ from its active site by Cu 2+ , PMI retains its structure with little changes in the TYCHO signal. Chapter 2: Phosphomannose Isomerase 2.3.9. Catalytic activities:

Diels-Alder cycloaddition

The Diels-Alder reaction is a cycloaddition of an alkene (dienophile) and a 1,3-diene with inter-and intramolecular forms being possible. [START_REF] Reetz | Artificial Metalloenzymes as Catalysts in Stereoselective Diels-Alder Reactions[END_REF] Because natural Diels-Alderases are rare, artificial Diels-Alderases are promising targets with a high potential for synthetic applications. it has been shown that a copper(II) is a good catalyst for this reaction. [START_REF] Matsuo | Artificial enzymes with protein scaffolds: Structural design and modification[END_REF] First, we decided to asssay the catalysis of the Diels Alders reaction of hallmark reactants: Cyclopentadiene (CP) and 2-azachalcone (scheme 13) by the artificial Cu-PMI and natural PMI because Zn is also a good Lewis acid, thus may catalyze this reaction. Nevertheless, large chelators such as 8-hydroxyquinoleine were reported to inhibit PMI by accessing its active site and specifically binding Zinc(II). [START_REF] Gracy | Studies on Phosphomannose Isomerase: II. Characterization As A Zinc Metalloenzyme[END_REF] Encouraged by a possible flexibility in the enzyme, the Diels-Alder cycloaddition of CP and 2-azachalcone was assayed but the reaction failed to proceed in the presence of either Zn-PMI or Cu-PMI.

Subsequently, the catalysis of the Diels-Alder cycloaddition of smaller commercially available substrates was aimed: Acrylonitrile and Furan. Here again, no Diels-Alder activity was observed (scheme 14) which could be explained by the fact that these are less reactive substrates. Therefore, two strategies were followed. The first aimed at further i) reducing the size of the substrates, while the second aimed at the ii) catalysis of a reaction that does not require substrate-access to the active site.

i) Design and synthesis of smaller Diels-Alder substrates Since PMI has a relatively small active site, three likely more suitable Diels-Alder substrates were designed. We designed structural analogs to the chelating motif of 2azachalcone i.e. two heteroatoms arranged in a pinch able to chelate Cu(II), linked to a vinyl group by a simple bond. We set our sights on compound 1 keeping in mind the primary amine resulting in the lack of delocalization of the vinyl double bond thus a lesser effect of the coordination on the reactivity of this dienophile (Scheme 15). We therefore designed a second analog, compound 3 closely related to 2-azachalcone in terms of possible electron delocalization. Because 3 still bears the chelating motif, we expect that it will be able to bind Cu(II) in a bidentate way, and because it is significantly smaller than azachalcone it should be able to enter the active site of PMI. Alcohol 2, is an intermediate in the synthesis of 3 and although it is a weaker ligand of Cu(II) (vs.

amines and carbonyls) and probably less reactive toward CP, it would be of interest in case the prospected artificial enzymes were highly efficient.

Oxidation reaction

In this second strategy, we proceeded to assay the possible peroxidase activity of Cu-PMI. Peroxidases are enzymes that catalyze the oxidation of a substrate by hydrogen peroxide or an organic peroxide as a catalyst. [START_REF] Everse | Heme Proteins[END_REF] Mostly, they are ferric heme proteins, but we can find also examples including superoxide dismutase containing copper [START_REF] Lewandowski | Inhibition of copper-zinc superoxide dismutase activity by selected environmental xenobiotics[END_REF] and even selenium-containing enzymes such as glutathione peroxidase. [START_REF] Everse | Heme Proteins[END_REF] Most peroxidases are devoid of substrate binding site. This catalytic reaction does not require substrate-access to the active site. The reaction proceeds by the oxidation of H2O2 at the metal cation in the active site. Then, the oxidized migrates outside the enzyme and react encountered substrates. The catalysis of the peroxidase activity of Cu-PMI was tested using H2O2 as oxidant and typical peroxidase co-substrates (co-S) such as 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 3,3',5,5'tétraméthylbenzidine (TMB) (scheme 16 and scheme 17). Oxidized TMB takes on a blue-green color whose absorbance can be measured at 370 nm or between 620 and 655 nm, while the absorbance of oxidized ABTS can be measured at 420 nm.

Preliminary results showed that the PMI-Cu artificial metalloenzyme does not catalyze the oxidation of TMB and ABTS. However, an important oxidation was obtained upon the addition of 60 equiv. of copper sulfate on the PMI-Cu enzyme. The binding of copper to the batch of apo-PMI was then evaluated by fluorescence titration.

Fluctuation of fluorescence was observed which is not comparable with those we reported previously. This indicates that the protein batch was damaged/contaminated.

The experiments will be repeated with a new batch that will be characterized by intrinsic fluorescence quenching and EPR beforehand.

CONCLUSION:

PMIs are metalloenzymes that catalyze the reversible isomerization between M6P and F6P. PMIs belong to the cupin superfamily of enzymes and their active site is located in the characteristic barrel of β-sheets. Artificial enzymes formed by incorporating transition metals or their complexes in β-sheets are rare and are poorly studied. Aiming at the preparation of a chimeric PMI-PLP protein, we prepared and characterized an artificial metalloprotein based on PMI. The enzyme expression vector yielded PMI comprising a His6 tag that we were able to cleave by introducing a TEVc/site using molecular biology techniques. Indeed, the expression plasmid was digested and a site for specific hydrolysis by acTEV protease was successfully introduced flanked by the sequence of the enzyme and that of the His6 tag. After purification on Ni-NTA column, the enzyme was separated from the tag by digestion with TEV protease. TYCHO showed that the thermal denaturation of PMI can occur at 60°C. PMI contains one zinc(II) cation per enzyme molecule and the removal of zinc(II) from the active site was performed by incubating the protein with 10 6 equiv. EDTA.

To prepare the artificial metalloenzyme, copper(II) was added and the successful transformation of PMI into an artificial copper(II)-containing enzyme was characterized by a relatively high affinity constant (KD = 3 µM). The EPR spectrum of PMI-Cu protein confirmed the presence of a single copper in the binding site of the enzyme. PMI-Cu protein complex did not catalyze Diels-Alder reaction. However, a peroxidase activity can be obtained by the artificial PMI-Cu enzyme after future works.

CHAPTER 3: PIRIN LIKE PROTEIN

INTRODUCTION

Following the studies that we carried out on PMI and described in chapter 2, we move on in this chapter to the second starting enzyme i.e. PLP. PLP is a bicupin containing an Fe 2+ in its active site. The characteristics of this protein will be described in details in section 3.2. Then, we will prepare and characterize a new artificial metalloprotein starting from the PLP of Pseudomonas aeruginosa by substitution of its native Fe 2+ by Cu 2+ .

In the following sections, the construction of the expression vector of PLP, that will be later used to express the chimeric PMI-PLP protein (Chimera 1) will be presented. The plasmid encoding for the His6-PLP that we obtained was revealed to cause coexpression problems thus, first we describe how we edited this plasmid to express only PLP. Second, the insertion of TEVc/site flanked by the His6-tag and the PLP sequences is shown. This site allows for the His6-tag to be cleaved after purification and before metal substitution. After further purification on a Ni-NTA column followed by the His6-Tag cleavage by TEV protease, the obtained protein was characterized by SDS PAGE electrophoresis and Western blotting. Its transformation into an artificial metalloprotein by exchanging the native Fe 2+ by Cu 2+ will then be presented including its characterization by various spectroscopic techniques.

STATE OF THE ART

In 1997, Winnacker et al. discovered a protein that they named it Pirin. [START_REF] Wendler | Identification of Pirin, a Novel Highly Conserved Nuclear Protein[END_REF] This non-heme iron protein was produced in every human tissue [START_REF] Wendler | Identification of Pirin, a Novel Highly Conserved Nuclear Protein[END_REF] and was highly conserved in prokaryotes, fungi, plants, and mammals. [START_REF] Soo | Pirin Regulates Pyruvate Catabolism by Interacting with the Pyruvate Dehydrogenase E1 Subunit and Modulating Pyruvate Dehydrogenase Activity[END_REF] According to biochemical studies, Pirin is a nuclear protein of 290 amino acids and its molecular weight is about 32 kDa. [START_REF] Wendler | Identification of Pirin, a Novel Highly Conserved Nuclear Protein[END_REF] Southern and Western Blots have shown that the Pirin genes' expression occurred in dot-like subnuclear cells structures for certain nuclear processes, such as the replication of DNA and/or the transcription of RNA polymerase II. [START_REF] Liu | Pirin is an iron-dependent redox regulator of NF-κB[END_REF] It has been shown that Pirin can be implicated in apoptosis and in cellular stress in eukaryotic organisms and in seed germination. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF][START_REF] Solomon | Copper Active Sites in Biology[END_REF] Studies have shown that Pirins and quercetinases have a lot in common structurally. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF][START_REF] Tranchimand | The rutin catabolic pathway with special emphasis on quercetinase[END_REF][START_REF] Fetzner | Ring-Cleaving Dioxygenases with a Cupin Fold[END_REF] Both of them belong to the cupin superfamily of proteins. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF][START_REF] Solomon | Copper Active Sites in Biology[END_REF] However, their metal ion needs are very different. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] It has been established that metal ions such as Cu 2+ Fe 2+ , and Ni 2+ were required for quercetinase activity. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF][START_REF] Nianios | Nickel quercetinase, a "promiscuous" metalloenzyme: metal incorporation and metal ligand substitution studies[END_REF] In 2013, it has been demonstrated that Pirin played a redox-sensing role in NF-kB regulation. [START_REF] Liu | Pirin is an iron-dependent redox regulator of NF-κB[END_REF] Only the ferric form of Pirin significantly enhanced the binding of NF-kB proteins to target kB genes. Though the ferrous and ferric Pirin differ only by one electron, their conformations was revealed to be different. [START_REF] Liu | Pirin is an iron-dependent redox regulator of NF-κB[END_REF] In particular, an R-shaped area forms the Pirin-NF-kB binding interface, which is responsible for the modulation of the NF-kB DNA binding 's properties. This area possesses 2 different conformations based on the identity and formal redox state of the metal. The Fe center is thus demonstrated to have an allosteric role in the binding of NF-kB proteins to the Pirin protein. [START_REF] Liu | Pirin is an iron-dependent redox regulator of NF-κB[END_REF] Pirin is thus proposed to act as a reversible functional switch that allows NF-kB to adapt to changes in the redox level at the cell nucleus. [START_REF] Liu | Pirin is an iron-dependent redox regulator of NF-κB[END_REF] Other studies have shown that Pirin-encoding orthologs were involved in a variety of biological activities, and the cellular function of this family of proteins is frequently linked to stress conditions. [START_REF] Talà | Pirin: A novel redox-sensitive modulator of primary and secondary metabolism in Streptomyces[END_REF] In 2018, Pirin was discovered overexpressed in surgical specimens from hormonesensitive prostate cancer (HSPC). This protein was therefore considered to be a potential novel therapeutic target for castration resistant prostate cancer. [START_REF] Arai | Pirin: a potential novel therapeutic target for castration-resistant prostate cancer regulated by miR-455-5p[END_REF] In 2015, Kataoka et al. searched for a protein classified into Pirin from Pseudomonas stutzeri zobelli which is a marine isolate strain used in agriculture, waste-water treatment and bioremediation. They planned to study the catalysis of oxidation reactions including quercetinase activity which has never been studied before in bioremediation. They found a protein that they called Pirin Like Protein (PLP). They Chapter 3: Pirin Like Protein cloned it, expressed it and determined its 3D structure by based of the crystal structure of Pirin (Figure 36). [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] The structure shows that PLP is indeed also a bicupine. PLP has a strong structural resemblance with Human Pirin protein as both proteins belong to the cupin superfamily which has two domains comprised of characteristic βstranded motifs and intervening loops. They both have a high degree of amino acid sequence homology of 39% (Figure 37). Hence, it was suggested that the PLP contains one Fe 2+ in the active site. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] The structure of PLP was predicted accurately by analogy to human Pirin protein by using Swiss model as seen by the heatmap in Figure 38. The biological role of PLP remains unclear as it is involved in a variety of biological processes. [START_REF] Talà | Pirin: A novel redox-sensitive modulator of primary and secondary metabolism in Streptomyces[END_REF] In 2018, Tala et al reported that the Pirin Like gene (PirA) was a unique redox-sensitive negative modulator of very long-chain acyl-CoA dehydrogenase, which catalyzes the beta-oxidation pathway in its first committed step. [START_REF] Talà | Pirin: A novel redox-sensitive modulator of primary and secondary metabolism in Streptomyces[END_REF] Furthermore, recent studies have shown that this protein exhibited quercetinase activities upon the incorporation of divalent metal cations such as Cu 2+ and Zn 2+ instead of iron. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] In the case of Cu 2+ , the holo-protein demonstrated the highest quercetinase activity as well as spectroscopic properties typical of type II Cu protein. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] Circular dichroism, EPR and absorption spectra of the Cu-PLP enzyme suggested that the coordination sphere contained His 59, His 61, His 103 and Glu 105 residues located in the consensus sequences of the cupin superfamily. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] In the present project, we have chosen this bicupin because its structure resembles that of the PMI. Indeed, the comparison of the proteins' structures has revealed a high structural resemblance between PMI and PLP: 45% of structure's identity and 68% of structure's similarity (Figure 40).

RESULTS AND DISCUSSION

3.3.1. Editing of the expression vector for the PLP:

The pUC18 vector which provided ampicillin resistance and the expression of His6-PLP was kindly provided to us by Professor Kataoka's team in Kanazawa University, Japan. [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] The plasmid was used to transform E. coli BL21(DE3) strain. Transformed bacteria were selected on ampicillin containing agar plates. In cultured colonies, the protein expression was induced by 0.2 mM IPTG as described in literature [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] and the expression of the His6-PLP was checked prior to purification. Anti His6-tag antibody western blot was performed on the bacterial pellets. Figure 41 shows the presence of a double band on the western blot suggesting the expression of two His tagged proteins of similar MW. It was impossible to separate these two proteins and to proceed then with the purification of His6-PLP on a Ni-NTA affinity column. Therefore, to avoid the expression of LacZa fused His6-PLP protein, we deleted the remaining portion of LacZa by PCR. The deletion was performed by designing forward and reverse primers that flank both sides of the region to be deleted (Figure 43). This deletion let to obtaining the desired recombinant protein that showed by western blot a single band at about 33 kDa corresponding to the expected MW of His6-PLP (Figure 45). The TEVc/site (5' GAAAACCTGTATTTTCAGGGC 3') was then inserted by PCR. The TEVc/site was flanked by the His6-tag and the PLP sequence so that after purification, the tag may be cleaved before metal substitution (Figure 46).

PLP

Then, in order to confirm the presence of the DNA sequence encoding for the His6 - PLP, plasmids were extracted, purified and sequencing was performed. Sequencing showed the expected sequences by confirming mutations at the desired locations, which was enough to confirm the PCR results. To obtain large quantities of the protein, the modified plasmid was used to transform E. coli BL21(DE3) cells. After the SDS-PAGE analysis, western blot was done and the protein bands were electro transferred horizontally onto a nitrocellulose membrane. The protein was detected using a specific primary antibody (Anti-His6-tag) followed by a secondary antibody labelled with peroxidase. Western blotting confirmed that the purified starting protein Chapter 3: Pirin Like Protein was indeed His-tagged (Figure 49). The eluted protein was dialyzed against a 50 mM MOPS buffer (50 mM MOPS and 300 mM NaCl), pH=7,6.

Iron (II) removal

PLP was transformed into an artificial metalloprotein by exchanging its iron(II) cation with a copper(II) cation. For this, the native iron cation was removed by EDTA and copper nitrate was then added. It was reported that the iron(II) cation of Pirin could be removed from the active site by incubation with 10 equiv. of EDTA overnight. [START_REF] Liu | Pirin is an iron-dependent redox regulator of NF-κB[END_REF] To completely strip the Fe 2+ of PLP, we investigated the effect of the addition of various amounts of EDTA at 4°C in 50 mM MOPS containing 300 mM NaCl, pH=7.6. A significant increase of the fluorescence of the PLP tag free was observed upon increasing the EDTA concentration indicating that removal of iron causes a change in the protein folding which leads to the increase of the fluorescence.

Low increase in fluorescence was observed with 10 equiv. of EDTA indicating that 10 equiv. of EDTA were not enough to remove the native Fe 2+ of PLP. Furthermore, adding Cu 2+ to the PLP tag free incubated with 10 equiv. of EDTA did not affect the intensity of the fluorescence (Figure 51). This suggests that Cu 2+ cannot replace the native Fe 2+ . A significant precipitation appeared upon the incubation with a concentration higher than 10 4 equiv. of EDTA. This precipitation suggested the denaturation of PLP that might be due either to the effect of EDTA on the PLP or to the stripped amount of Fe 2+ from the PLP's active site. The denaturation of the PLP was verified by TYCHO which Chapter 3: Pirin Like Protein verifies the structural integrity of the protein and determines the inflection temperature. Figure 52 showed that the native PLP folding is maintained at increasing temperature and its thermal denaturation can occur only at 75°C. Upon the addition of 10 4 equiv. of EDTA, PLP's signal changed, suggesting the change in its folding. Upon the addition of 10 3 equiv. of EDTA, the PLP retains its structure with little changes in the TYCHO signal and its thermal denaturation can occur at 75°C (Figure 53). Similarly, quenching of the fluorescence was also observe (see below 3.3.4.2). Hence, to achieve the complete removal of iron(II) we discovered that 10 3 equiv. of EDTA were required followed by dialysis for 2 days.

Evolution of fluorescence in the presence of metals:

To study the changes in the folding of the PLP, we investigated the interaction of the protein with different metal cations by fluorescence spectroscopy.

Since the His6-tag is an excellent ligand for metal cations, [START_REF] Porath | Immobilized metal ion affinity chromatography[END_REF] the added metals bind first the His6-tag and then can bind to other amino acids. For this, the tag should be cleaved before the fluorescence titration for trustworthy results.

We studied the fluorescence titration of the PLP tag free by different metals before removing the native iron cation by EDTA. Figure 54 shows a non-significant variation of the fluorescence following the addition of both Copper sulfate and Zinc sulfate. This suggested that Copper and Zinc could not bind to the protein, which confirmed the presence of Fe 2+ in the active site of the PLP preventing the binding of both metals. As for Figure 55, a significant fluctuation of the fluorescence could be observed upon the addition of increasing amounts of nickel nitrate. This could be due to non-specific binding of nickel to PLP with low affinity. Chapter 3: Pirin Like Protein Since PLP is suspected to contain Fe 2+ in its active site, it was necessary to remove this metal cation before transforming PLP into an artificial metalloenzyme. For this, Fe 2+

was removed by incubating PLP with 1000 equiv. of EDTA for 8 hours at 4°C. Then, EDTA was removed by dialysis in 50 mM MOPS, 300 mM NaCl, pH=7.6 for two days.

The iron free His6-tag free protein could then be reconstructed by binding copper(II).

Upon addition of increasing amounts of Cu(II) (0.2-55 equivalents) to the iron free PLP, the intensity of the protein intrinsic fluorescence signal decreased drastically (Figure 56). The insertion of other metal ions such as Zn 2+ (Figure 58) and Ni 2+ (Figure 59) has also been studied by fluorescence spectroscopy. Similarly to the titration using copper(II), a quenching of the fluorescence of PLP was also observed. Chapter 3: Pirin Like Protein To compare the affinity of each metal for PLP, we present in Figure 60 the variations of the fluorescence of PLP at 320 nm upon addition of increasing concentrations of the various metal cations. The lowest fluorescence quenching was observed in the case of Zn 2+ which indicated that there was a weak interaction between Zn 2+ and PLP (Figure 60). A slightly higher variation in fluorescence was observed in the case of Ni 2+ which indicated that there was a better interaction between Ni 2+ and PLP (Figure 60). Chapter 3: Pirin Like Protein Nevertheless, we noticed the presence of two inflexion points that could be due to non-specific binding of nickel to the protein. Finally, the higher variation in fluorescence was observed in the case of Cu(II) which indicated that this metal cation had the best interaction with PLP (Figure 60). Similarly to the PLP-Cu complex, the PLP-Ni concentration was plotted against the total added Nickel concentration and the resulting curve (Figure 61) was fitted by using the same equation as above, which allowed the determination of the KD value for the PLP-Ni(II) complex. A good fit was achieved with n=1 and yielded KD = 0.7 µM. However, a better fit was obtained with n= 2 and yielded KD1 = 0.5 and KD2 = 2.1 µM. This indicates that the PLP can possess more than one binding site for Ni 2+ .

The titration thus confirmed the highest affinity of PLP for copper. Furthermore, it confirmed the formation of 1/1 copper/protein complex characterized by a KD of 0,5 µM. Since the PMI showed an increase in fluorescence following the addition of copper, unlike the PLP which showed a decrease, it was necessary to find an alternative method to study the copper affinity for the protein that could be further used to characterize the chimera. Thus, another technique, isothermal titration calorimetry ITC, was used to confirm the fluorescence results as described in the paragraph below.

A B Chapter 3: Pirin Like Protein

Isothermal titration calorimetry (ITC)

Isothermal titration calorimetry (ITC) is one of the best methods used to determine the thermodynamic parameters of interactions in solution. [START_REF] Barbosa | Synthesis and ITC characterization of novel nanoparticles constituted by poly(-benzyl L-glutamate)-cyclodextrin[END_REF] It is most often used to study the binding of small molecules (such as medicinal compounds) to larger macromolecules (proteins, DNA etc.). It is based on the concept that heat can be either absorbed or generated during the process. At constant temperature, a solution containing a ligand is added to a cell containing a solution of the macromolecule. The interaction between the two partners then results in heat release or absorption. [START_REF] Barbosa | Synthesis and ITC characterization of novel nanoparticles constituted by poly(-benzyl L-glutamate)-cyclodextrin[END_REF] The binding of copper(II), zinc(II) and Ni(II) to metal-free PLP was first studied without adding EDTA. As shown in Figure 62, Figure 63 and Figure 64, when metal cations were added into the native PLP, no heat exchange was found during these three experiments. As for fluorescence titration, this result proved that there is no interaction between PLP and any metal before stripping the iron from its active site. To completely remove the Fe(II) cation of the PLP, 10 3 equiv. EDTA were added. As shown in Figure 65, no heat exchange was observed when Zinc sulfate was added into the iron free PLP. This result shows that there is no interaction between PLP iron free and Zn(II) which corroborates the results obtained by fluorescence titration. However, when copper(II) was injected into a solution of iron free PLP, no exothermic reaction was observed. As for Figure 66, it is not possible to reliably determine any thermodynamic parameter. This may be due to the fact that this method was not suitable to determine the affinity of copper into PLP or the protein could bind to the alloy of the ITC cell preventing the binding of the added metal cations.

Furthermore, we can suggest that only a part of the protein can bind the metal which can be due to the EDTA effect of the PLP's folding. However, when nickel(II) was injected into a solution of iron free PLP, an exothermic reaction was observed after the first few injections. Figure 67 shows a typical example of the curve that was obtained after integration of heat signal. However, for the PLP-Ni complex, a 0,3 Ni(II)/PLP stoichiometry could be determined which could be the sign of the presence of multiple binding sites for Nickel in PLP (Figure 67) or denatured protein.

These results confirm the existence of interactions between Ni 2+ and PLP. Consequently, the Kd PLP-Ni were determined from ITC measurements and were equal to 0,6 µM. After ITC experiments, the samples coming out of the titrations were analyzed by TYCHO to check the integrity of the protein fold. Figure 68 shows a little difference in the signal. This confirms that the protein retains its folding upon the EDTA addition followed by the addition of different metals. The thermal denaturation of these proteins occurs around 75°C. However, the different ITC results were not reproducible when the PLP remained longer time without any metal and the stoichiometry was changing. This confirmed that the metal of PLP conferred a structural stability to the protein.

Hence, based on the last ITC results:

-ITC is not a suitable method to analyze the binding of Cu 2+ in PLP.

-The ITC cells are made of metal alloys thus PLP can bind to the cell preventing the binding of the added metal cations.

-The folding problems of PLP can be due to two reasons: i) EDTA's can affect the protein's folding when added in high concentration.

It is noteworthy that this chelator is known to have high affinity for binding metalloproteins, destabilizing their structure and forming aggregates in solution. [START_REF] Janecki | Denaturation of metalloproteins with EDTA to facilitate enzymatic digestion and mass fingerprinting[END_REF][START_REF] Müller | pH-dependent formation of ethylenediaminetetraacetic acid supramolecular aggregates[END_REF] ii)

The absence of the iron cation presented in the PLP's active center conferring a structural role which can alter the PLP's folding. Chapter 3: Pirin Like Protein

Electron paramagnetic resonance EPR:

The EPR spectrum of the metal free PLP and PLP-Cu complex were measured at 10 K at a frequency of 9.3 GHz. As expected, the iron free PLP did not show any EPR signal, which confirmed that the active site was free of iron(III) after incubation with EDTA.

However, the EPR spectrum of the PLP-Cu complex showed a quadruplet signal centered at g// = 2.06, g⊥= 2.32 values, hyperfine coupling constants of A// = 18.6 mT which confirmed the presence of a single type of copper(II) in the binding site (Figure 69).

Effect of high concentrations of copper on the PLP:

To investigate the effect of the addition of copper to iron free PLP, we studied the protein's structural folding of the protein as a function of the increasing amounts of copper sulfate by TYCHO.

Upon the addition of 2 equiv. of Cu(II), the PLP retains its folding (Figure 70). However, upon the addition of 10 equiv. of Cu(II), the folding profile of PLP-Cu protein complex changed (Figure 71). This result indicates that high concentrations of copper can alter PLP's folding. Chapter 3: Pirin Like Protein

CONCLUSION:

Pirin Like protein (PLP) is a metalloenzyme that resembles PMI. It belongs to the cupin superfamily of enzymes, and their active sites are located in the characteristic barrel of β-sheets. The biological role of PLP remains unclear. A previous study has shown that PLP could be transformed into an artificial metalloenzyme by substituting its iron cation by a divalent metal cation such as Cu 2+ . Aiming at the preparation of a chimeric PMI-PLP protein, we first prepared and characterized an artificial metalloprotein based on PLP. The enzyme expression vector yielded PLP comprising a His6-tag co-expressed with second protein formed of a portion of LacZ fused to His6-PLP. The coexpression was confirmed by an anti His6 western blot which revealed two bands instead of a single band. To avoid this coexpression, the expression plasmid was edited by deleting the remaining portion of LacZ. The expression plasmid was digested and a site for specific hydrolysis by TEV protease was successfully introduced flanked by the sequences of the enzyme and that of the His6-tag. Purified and sequenced plasmid DNA containing the insert sequence was used to transform BL21(DE3) for expressing the protein. After expression and purification on an Ni-NTA column, the enzyme was separated from the His6-tag by digestion with TEV protease. Following the digestion, the TEV protease with its polyhistidine tag was easily removed from the cleavage reaction by purification on a Ni-NTA affinity column. The produced PLP contains one Fe(II) cation per enzyme molecule, therefore the removal of Fe(II) from the active site was performed by incubating the protein with 10 3 equiv. EDTA. To prepare the artificial metalloprotein, Ni 2+ , Zn 2+ and Cu 2+ were added to the iron free protein. A relatively high affinity constant (KD = 0.5 µM) was measured by fluorescence quenching upon addition of increasing amounts of copper sulfate. The EPR spectrum of the PLP-Cu complex confirmed the presence of a single copper species. TYCHO results showed that the thermal denaturation occurs at 75°C and high concentrations of EDTA and copper sulfate can alter the folding of PLP.

Hence, PLP was successfully transformed into an artificial copper(II)-containing enzyme. Finally, the Diels-Alder activity of the Cu-PLP enzyme can now be examined.

CHAPTER 4: CHIMERIC ENZYMES

INTRODUCTION:

As mentioned in chapter 1 section 1.2, artificial fragments of DNA can now be easily synthesized and subsequently used to express synthetic proteins such as chimeric enzymes.

Chimeric enzymes have been found useful for a wide range of applications including in medicine, chemistry, and agriculture. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Therefore, we designed and followed a new strategy to develop a chimeric PMI-PLP enzyme. Such an enzyme contains two neighboring active sites and should be able to catalyze cascades of reactions.

To design fusion enzymes such as chimeric enzymes, it is especially important to start from partner proteins that are active under similar conditions such as temperatures and pH profiles, as mismatched parameters could affect the overall performance. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] In this project, we therefore chose two enzymes of the Cupin family i.e. PMI and PLP which have high structural similarities. They are both bicupins characterized by a structure formed of two beta barrel domains, each possessing one active site. To construct the PMI-PLP bicupine chimera, the inter-domain of the two metal-containing cupin subunits of the two original bicupins will be fused.

In this chapter, we will discuss the followed strategy to design chimeric enzymes by combining catalytic domains of PMI and PLP. Structural modeling studies, determination of the suitable linking point and molecular biology techniques used for ensuring the successful expression of the enzymes will all be discussed. Furthermore, all the difficulties that we have faced in the construction as well in the purification of the chimeric proteins will be presented.

RESULTS AND DISCUSSION

Structural modeling

As stated earlier, the chimeric enzyme will be designed by combining the catalytic domains of PMI and PLP (i.e. the C-terminal domain of the PMI and the N-terminal domain of PLP respectively). For this, we searched for a suitable linking point that is featured in PMI and PLP but is neither involved the active sites nor in the overall folding of the proteins. (Scheme 18). Hence, we started searching where there are little interactions between residues in both proteins. For this, we used the web interface elNémo to simulate the low frequency normal modes of vibration of PMI and of PLP. Note that, elNémo can perform calculations for all-atom systems and provides simple visualized results. [START_REF] Studier | Use of T7 RNA polymerase to direct expression of cloned genes[END_REF][START_REF] Sahdev | Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies[END_REF] Chapter 4: Chimeric enzymes Figure 72 shows the sequence of frames of the video providing calculations resulting by elNémo. Upon providing elNémo with the crystal structure of PMI (pdb number 1PMI) the visual inspection of the first, second and third normal modes of vibration show that the two cupin domains vibrate independently (Figure 72). This suggests that it is reasonable to associate different cupin domains in a chimeric enzyme. The third normal mode of vibration originates at the center of the linker of the cupin domains.

At high intensity this vibration leads to the dissociation of the linker at its center. It is thus reasonable to assume that center of the linker is a suitable association point for the different cupin domains. For PMI, the center of the vibration in the interdomain linker is Met357. For PLP, same procedure was followed to simulate the low frequency normal modes of vibration. Similar studies showed that in the case of PLP, the center of the third normal mode of vibration is Ala138.

Based on these results, the chimera was envisioned to be formed of PMI amino acids Met1 to Met335 fused to PLP amino acids Gly12-Ala138.

Then, we proceeded to the structural modeling of the envisioned chimera using two different molecular modeling servers dedicated for protein structures prediction based on known structures of proteins of similarities sequences: MODELLER and I-TASSER.

As we mentioned before in section 1.2.3.1, MODELLER automatically calculates a model by aligning a sequence with known related structures [START_REF] Leman | Macromolecular modeling and design in Rosetta: recent methods and frameworks[END_REF][START_REF] Ghosh | Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli[END_REF] whereas I-TASSER predicts a 3D structural model based on aa sequences. [START_REF] Swartz | Advances in Escherichia coli production of therapeutic proteins[END_REF] For this, the structural models of the chimeric PMI-PLP protein were predicted by I-TASSER according to its amino acid sequences (Figure 73). Figure 73 shows that the overall chimeric PMI-PLP protein superposes well with each of the two starting enzymes. More careful inspections revealed that molecular modeling using I-TASSER allowed to predict a 3D structure of the chimeric enzyme presenting a correct folding (Figure 74).

On the contrary, molecular modeling using MODELLER led to a structure with a helix from the PMI fragment (in red) clashing with the cupin domain of the PLP fragment (in yellow). To avoid this possible problem, a second chimera called thereafter ''Chimera 2'' was designed. In this case, the PMI loop responsible for the clash was replaced by the analogous loop from PLP (loop from the second cupin domain of PLP) which is much smaller. Fortunately, the modeling of this new structure by both I-TASSER and MODELLER indicated that Chimera 2 should fold correctly (Figure 75).

Design and construction of the expression vector of the first chimeric PMI-PLP protein:

We examined the DNA sequence of the starting proteins that we wanted to amplify and we designed the primers that include restriction sites facilitating the fusion and are suitable PCR amplification of the fragments. The steps followed to construct the expression vector encoding for the Chimera 1 are illustrated below (scheme 19 and scheme 20). Then, the sequence encoding for the PMI fragment was amplified and Nde1 and Fse1 restriction sites were inserted. The former was planned for inserting the sequence in the expression vector. The latter was introduced to be used to fuse the two amplified fragments. The PLP fragment was also amplified and BamH1 and Fse1 restriction sites were inserted for the same reason as earlier. The presence of the PCR amplicon and its size were determined by electrophoresis alongside a DNA size marker on a precast 1.2 % agarose gel (Figure 76). Figure 76 shows bands around 1300 bp and 400 bp corresponding respectively to the molecular weight of PMI and PLP fragments. The two PCR products were then purified. Chapter 4: Chimeric enzymes Subsequently, DpnI i.e. a restriction enzyme that possesses a recognition sites that is methylated, was added to the mix to catalyze the hydrolysis of the original PMI and PLP expression vectors. Transformation resulted in bacteria that contained the desired plasmid as demonstrated by specific PCR amplifications.

First, we verified the presence or absence of DNA insert in plasmid constructs by Colony PCR. Then, in order to confirm the presence of the DNA sequence encoding for the Chimera 1, sequencing was done at Eurofins using the Sanger's method. It showed the expected sequence. E.coli BL21 (DE3) strain was used for expressing the protein on agarose/ampicillin plates.

Expression and purification of the chimeric PMI-PLP enzyme:

To obtain substantial quantities of protein, the modified plasmid was used to transform E. coli BL21(DE3) strain. A pre-inoculum from a single colony was grown in a starter LBampicillin (100 μg/mL) culture, at 37 °C for 18 hours. Terrific Broth (TB) supplemented with ampicillin (50 μg/mL) was inoculated from the preinocculum at 1/100 th and grown at 37 °C until the OD600 reached a value between 0.6 and 0.8. At this stage, either 0, 0.1, 0.2, or 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and incubation was continued at 20 °C for 18 h.

The expression level of the induced protein was checked by SDS PAGE electrophoresis followed by Coomassie blue staining. For this, bacterial cells were pelleted and resuspended with sterile water. As shown in Figure 78, no band was observed without IPTG addition. On the contrary, a band of about 55 kDa corresponding to the molecular weight of the His6 chimeric enzyme was observed upon addition of 0.1 mM IPTG or more. Hence, this 0.1 mM IPTG is enough to induce the production of the first chimeric PMI-PLP protein. In parallel, anti His6-tag antibody western blot was performed on the bacterial pellets. It confirms that the produced induced protein was indeed His tagged (Figure 79). The purification was monitored by SDS-PAGE electrophoresis followed by Coomassie blue staining that revealed a band at a molecular weight of about 55 kDa (Lanes 7, 8 and 9 corresponding to this protein). Unfortunately, other bands were also present thus the tagged protein was not pure (Figure 80A). In parallel, western blotting using anti His-tag antibody confirmed its identity (lanes 7,8 and 9, Figure 80B). The fraction containing the tagged protein were combined and the protein concentration measured. Only 1 g per liter of culture were measured. This result indicates that only a small quantity of the over expressed protein observed after induction with 0.1 mM IPTG (Figure 78) was not found in the soluble protein fraction A) B) Chapter 4: Chimeric enzymes that was used for purification. The low purification quality was thus due to low quantity of soluble chimera.

In addition, we assayed to optimize the separation conditions by changing the imidazole concentration in order to reduce the binding of contaminant proteins, and thus increase the final purity. Nevertheless, a TEV protease cleavage followed by a purification on His Trap Column were done but the purification remained unsuccessful.

Several strategies were followed to attempt to favor the solubilization of the protein such as:

-Changing the bacterial strain and using E. coli BL21(DE3) pLysS cells which allow high efficiency protein expression, express T7 RNA polymerase which can improve the expression and also encode T7 lysozyme that suppresses basal expression of toxic target proteins prior to induction. This can prevent toxicity problems when adding IPTG. -Variation of the induction temperature and culture time to optimize the chimera's expression (37°C for 4 hours, 6 hours and 8 hours, 30°C for 6 hours).

Nevertheless, it has been known that usually low temperatures can improve the protein solubility. [START_REF] Sahdev | Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies[END_REF] Hence, 37 °C gives faster growth and usually higher levels of production for proteins that don't tend to form insoluble inclusion bodies.

-Cool down the culture by placing it on ice before induction with IPTG aiming at decreasing the rate of protein synthesis, especially if the chimera is toxic for E.coli proteins.

Unfortunately, despite all these attempts, the soluble fraction remained low and the purification not successful. Because folding problems were suspected for chimera 1 (see section 4.2.1), we did not explore further options for its solubilization. Instead, we proceeded by using chimera 2.

4.2.4.

Design and Construction of the expression vector of the Chimera 2:

The sequence encoding for chimera 2 was purchased. As we mentioned before, Chimera 2 is similar to Chimera 1 with the difference that a PMI loop suspected to clash with a cupin barrel in chimera 1 was replaced by a much smaller loop from PLP in chimera 2 (Figure 81). Identical aa are in black, similar aa are in blue and different aa are in red.

The steps followed to construct the expression vector encoding for the Chimera 2 are illustrated below (scheme 21).

The pET-28b plasmid known for its high efficiency for the expression of proteins was chosen as a vector. This vector is able to reduce leaky expression of proteins which is under the control of T7 lac promoter to promote tightly and strong gene expression. [START_REF] Studier | Use of T7 RNA polymerase to direct expression of cloned genes[END_REF][START_REF] Dubendorf | Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor[END_REF] This vector encoding for the Small Ubiquitin-like Modifier (SUMO) tag protein was double digested by BamHI and PstI and then purified. The result of the digestion of the vector was analyzed by agarose gel electrophoresis and ethidium bromide staining (Figure 82). A single band around 6 Kb was visible after the digestion which confirmed the digestion of the plasmid. The undigested plasmid bands appeared at three different levels in the gel which can be assigned to the different tertiary structures of circular plasmid DNA (Supercoiled, nicked and relaxed). Chapter 4: Chimeric enzymes The digested plasmid was extracted from the gel as described in materials and methods. In parallel, the synthetized sequence of chimera 2 was amplified by PCR, which also allowed the insertion of restriction sites BamhI and PstI. The amplification was followed by a double digestion by BamhI and PstI. The result of this digestion was purified and analyzed by agarose gel electrophoresis and ETBr. The two purified products extracted from the gel could anneal. Excess of the digested double stranded DNA insert encoding for the chimera 2 was added to the digested pET-28b plasmid and the mixture ligated using instant DNA ligase. The ligation mixture was used to transform DH5alfa competent bacteria. A pre-inoculum from a single colony was grown in a starter culture LB supplemented with 75 µg/mL Kanamycin and cells were grown overnight. Then, the plasmid was extracted and its sequence was verified by sequencing. It showed the expected sequence.

Expression of Chimera 2:

To obtain large quantities of protein, the sequenced purified plasmid DNA containing the insert sequence was used to transform BL21(DE3) that were then selected on agar/kanamycin. A pre-inoculum from a single colony was grown in a starter culture YT-Kanamycin. YT supplemented with Kanamycin was inoculated from the preinocculum at 1/100 th and grown at 37 °C until the OD600 reached a value between 0.6 and 0.8. Different concentrations of IPTG were then added and incubation was continued at 20 °C for 18 h. Bacterial cell were pelleted and kept at -20 °C. Analysis of supernatant fractions and pellets was done by SDS-PAGE followed by Coomassie blue staining (Figure 83).

Upon five hours of induction, a band with the same intensity (Figure 83 Before proceeding to the first purification, we tested the solubility of this protein. For this, BL21 cells, transformed with the plasmid containing the DNA encoding for chimera 2, were grown either in LB or in TB medium. The produced protein was then analyzed on SDS PAGE gel before and after the cell lysis in order to verify its solubility in the cells.

As shown in Figure 84, before lysis, Chimera 2 is well expressed in cells. First, in absence of induction, in LB medium, no band characteristic of this protein was observed after Coomassie blue staining (lane 1). On the contrary, after IPTG induction, a band of high intensity at MW of about 60 kDa was observed in both media (lanes 2, 3 and 4 for the LB medium, lanes 6, 7, 8 for the TB medium). In the case of TB, induction was observed without IPTG addition (lane 5), as it is often the case with this auto-inducing medium. After cell lysis, SDS-PAGE analysis followed by Coomassie blue staining of the various insoluble fractions (inf) and soluble supernatant fractions (sf) was performed (Figure 85). Without IPTG induction in LB medium, the chimeric protein was not found in the soluble fraction (Lanes 1 and 2). However, upon induction by 0,1 mM IPTG a band with a high intensity, at a molecular weight of about 60 kDa corresponding to that of chimera 2 could be observed in lanes 4, 6 and 8 (induction with 100 µM, 200 µM and 400 µM IPTG respectively), thus chimera 2 was found only in the lysate most probably in inclusion bodies. Such a band could not be seen in lanes 3,5 and 7 corresponding to the soluble fractions. Hence, this problem may be due to different reasons such as:

-Chimera 2 can be a toxic protein.

-Chimera 2 does not fold correctly.

This was unexpected since, as mentioned before in section 4.1, the structural modeling using both MODELLER and I-TASSER indicated that Chimera 2 should fold correctly. A recent new prediction of the structure of chimera 2 using Rosetta [START_REF] Leman | Macromolecular modeling and design in Rosetta: recent methods and frameworks[END_REF] confirmed that it should fold correctly.

-Solubility problems deriving from those of the starting enzymes:

To check this point, we moved to verify the solubility of the two PMI and PLP starting enzymes. The same procedure was followed as for the Chimera 2 and SDS-PAGE electrophoresis followed by Coomassie blue staining (Figure 86) as well as Western Blot (Figure 87) of supernatant fractions and pellets after the lysis of the expressed proteins were done.

A comparison of the SDS-PAGE analysis with the Western blot using anti His6-tag antibody shows that the PMI is indeed expressed in the supernatant, as a band at a Chapter 4: Chimeric enzymes MW of about 50 kDa was observed in lane SPMI, which corresponds to the molecular weight of His6-PMI.

On the other hand, it appears that PLP is expressed in both supernatant and inclusion bodies. Indeed, a band at MW of about 33 kDa was observed in both PPLP and SPLP lanes.

Concerning the chimeras, bands were observed in lanes PCh1 and PCh2 at MW corresponding to the expected MW respectively for Chimera 1 (50 kDa) and Chimera 2 (60 kDa) which suggests that the two proteins are mainly expressed in the inclusion bodies rather than the supernatant. Thus, this can be due to the expression of PLP in both supernatant and inclusion bodies. Therefore, Figure 87 suggests that the Chimera 2 appears to be better expressed (PCh2) than the first chimeric PMI-PLP protein (PCh1). Several means can be envisioned to enhance the expression of a protein under a soluble form including:

i)

The change of the expression vector by using weak promoters. [START_REF] Ghosh | Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli[END_REF][START_REF] Swartz | Advances in Escherichia coli production of therapeutic proteins[END_REF][START_REF] Firat Duzenli | Promoter Bibliography engineering for the recombinant protein production in prokaryotic systems[END_REF] It has been know that regulating the gene's expression is one of the most important processes in protein production. [START_REF] Firat Duzenli | Promoter Bibliography engineering for the recombinant protein production in prokaryotic systems[END_REF] Therefore, promoters are one of the most important regulatory components in a host cell since they may directly influence the degree of recombinant gene expression. [START_REF] Firat Duzenli | Promoter Bibliography engineering for the recombinant protein production in prokaryotic systems[END_REF] ii)

The change of the host strain to avoid possible missing post translational modifications or incorrect disulfide formation. [START_REF] Ghosh | Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli[END_REF]100 It has been shown that protein's solubility can be improved upon osmotic induction with high salt as NaCl. 100 iii)

The coexpression of the protein with chaperones that aid its correct folding [START_REF] Ghosh | Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli[END_REF]101 and in some cases, leads to enhance the production of active proteins. 102,103 The solubilization of the protein out of inclusion bodies (denaturation of the protein followed by renaturation) can also be envisioned especially in this case as the structure of the refolded protein is expected via molecular modeling and the starting proteins PMI and PLP. 101 Hence, to generate a soluble chimeric protein, we proceeded with the coexpression of chaperones. The latter help in folding other proteins and maintaining the proteostasis in cells, which prevents the formation of aggregates and thus inclusion bodies. 102,103 For this, competent cells containing a pG-KJE6 plasmid 102 were employed as it encodes for DnaK-DnaJ-GrpE and GroEL-GroES chaperone teams 102 (Figure 88). These two In contrast, no bands were observed in lanes 1, 3, 5 and 7 corresponding to the supernatant fractions (Figure 89).

CONCLUSION:

We have studied here an interesting new strategy that aims to develop an artificial chimeric metalloenzyme that possesses multiple active sites by the fusion of catalytic domains of 2 related enzymes: PMI and PLP.

First, the strategy we followed to prepare the PMI-PLP bicupin chimera was based on structural modeling studies and on the determination of the linking point between the two catalytic domains of PMI and PLP. elNémo was used to simulate to simulate the low frequency normal modes of vibration of PMI and of PLP. The movements of all atoms/domains in PMI and PLP were thus studied and visualized. The 1 st and the 2 nd mode of vibration of PMI show that the 2 cupin domains vibrate independently. The 3rd normal mode of vibration originates at the center of the linker of the cupin domains. So, the association of the two different cupin domains can occur by fusion at the center of their interdomain linkers.

The sequences encoding for the PMI active site and the PLP active site were cloned by PCR. PCR products were then purified, annealed and ligated via the Gibson Assembly method. Hence, the expression vector containing the DNA encoding for the PMI-PLP chimera was constructed, and the chimera was successfully expressed in E.coli BL21 but unfortunately the first purification was not satisfying.

Since the obtained protein was not pure, we tried to optimize the purification via different methods. Since molecular modelling using MODELLER revealed a possible clash between the  helixes in the variable region deriving from PMI and the barrel deriving from PLP, a second chimeric enzyme, Chimera 2, was constructed by replacing the PMI loop responsible for the clash by the analog PLP loop which is much smaller.

Chimera 2 was expressed better than the first chimera but unfortunately, the expressed protein was found in the insoluble fractions, most probably in inclusion bodies. The coexpression of chaperones proteins with chimera 2 failed in solubilization the protein.

As perspectives, alternative methods such as the use of denaturing agents will be examined to solubilize the inclusion bodies. Hence, the protein will be denatured which will require its refolding. 106,107 The correct folding of the chimera could be verified by comparison with that of the parental proteins.

In the future, this chimeric enzyme could be used to catalyze different reactions and different cascade reactions.

CHAPTER 5: HALF PROTEINS

INTRODUCTION

As we mentioned before, PMI and PLP are so-called bicupines because they are formed of two cupin domains composed each of one barrel of four β sheets. These cupin domains are connected by an inter-domain consisting of an α helixes.

We decided to study the effect of the loss of the first cupin domain on the second one, which contains the active site. For this, each half of the 2 starting enzymes (Half PMI and Half PLP) made of one cupin domain which contains the active site were express and purified. The aim was then to evaluate the maintenance of the fold and the initial activity of the protein in the absence of the second domain.

In this chapter, we will describe the cloning by molecular biology techniques of the two half enzymes, half-PMI and half PLP and their characterization by SDS Page electrophoresis and Western blot.

The construction of the expression vector encoding for the His6-tagged half PMI as well as for the His6-tagged half PLP will first be described, followed by the cloning and expression of these proteins in E. coli.

After purifications on a Ni-NTA column, the characterization of the obtained proteins by SDS PAGE electrophoresis and Western blot will then be shown.

RESULTS AND DISCUSSION

Half PMI protein

Construction of the expression vector for Half PMI

To construct the expression vector for Half PMI, we used the enzyme expression vector (pRSET-C vector) encoding for the PMI comprising a His6 tag as well as a TEV protease cleavage site. This vector was described in Chapter 3 and was used to express both PMI and Chimera 1.

The target DNA sequence encoding for the His6 tagged PMI's active site and His6 tag flanking TEVc/site was examined and the suitable primers to be used to bind and allow its replication by PCR were designed. The designed primers facilitated the amplification of the first cupin domain of PMI, TEVc/site, His6 tag and the pRSET-C vector including BamH1 restriction site. Additionally, the primers allow the insertion of BamH1 restriction site at the other end (Scheme 22). This amplification resulted in the deletion of the DNA sequence encoding for the cupin domain that does not contain the active site. The amplified samples were analyzed by electrophoresis/Syber safe staining and purified.

The purified product was then digested by BamH1 restriction enzyme generating two sticky ends with unpaired bases at the end of the fragments. Subsequently, DpnI was added to the mix to catalyze the hydrolysis of the original PMI expression vector.

Digestion was followed by ligation using instant sticky ligase master mix kit. The ligation mixture was used to transform E. coli (DH5alfa) strain. Transformed cells were suspended into LB medium supplemented with 100 ug/ml Ampicillin. In order to confirm the DNA sequence of the construct, a colony of the transformed and selected bacteria was grown and plasmids were extracted, purified and sequenced. They showed the expected sequence.

Expression and purification of the His6-Half PMI

E. coli BL21(DE3) cells were used for expressing the protein. The purified and sequenced plasmid DNA was used to transform BL21(DE3) cells that were then selected on agarose/ampicillin plates.

A pre-inoculum from a single colony was grown in a starter culture in LB-ampicillin medium. TB supplemented with ampicillin was inoculated from the preinoculum at 1/100 and grown at 37 °C until the OD at 600 nm reached a value between 0.6 and 0.8. 0.1 mM IPTG was then added and incubation was continued at 20 °C for 18 h. Bacterial cells were pelleted and kept at -20 °C. To lyse the cells prior to the purification, the bacterial pellets were resuspended in the lysis buffer in the presence of benzonase and lysozyme. The mixture was then sonicated and centrifuged to eliminate all solids. The clear solution containing the His6 tagged protein was purified on an Ni-NTA agarose column. The various eluted fractions were analyzed by SDS-PAGE followed by Coomassie blue staining. As shown in Figure 91, the elution fractions show different bands revealing a non-pure protein. However, the corresponding molecular weight of the Half PMI was expected to be about 40 kDa. No band with high intensity was observed in the elution fractions corresponding to this molecular weight. This result indicates that the protein is not well expressed. To optimize the expression of the protein, another bacterial strain was used. Purified and sequenced plasmid DNA encoding for Half PMI was used to transform BL21(DE3) pLysS (see Chapter 2 PMI) that were then selected on agarose/ampicillin. The same procedure as above was followed to express and purify the protein on a Ni-NTA agarose column. As shown in Figure 92, the elution fraction shows different bands than those observed above: A band at a molecular weight of about 40 kDa corresponding to this protein was observed, other bands were also present thus the protein was not pure. Despite this attempt, the purification was not successful.

Figure 92: SDS-PAGE electrophoresis followed by Coomassie blue staining of the elution fractions collected after the purification on an Ni-NTA agarose column of His6-Half PMI using the BL21 E.coli pLysS strain. Chapter 5: Half proteins A western blot using anti His6-tag antibody was done. Figure 93 shows three bands corresponding to the fractions eluted with buffer containing 300mM imidazole. This suggests that the elution fractions contained different His6 tagged proteins: a band with high intensity corresponding to a protein with a molecular weight of about 40 kDa corresponds to the expected MW of His6-Half PMI. This confirms that the protein was indeed His6 tagged. Two other bands may correspond to other histidine rich proteins that could bind the Ni-NTA column and weakly bind to the His6-tag antibody. Hence, folding problems were suspected and the low purification quality can be due to low quantity of soluble His6-Half PMI that can be found in inclusion bodies.

Half PLP protein

Construction of the expression vector for Half PLP

Starting from the full PLP expression vector, the objective was to extract the sequence encoding for the active site-containing cupin domain of PLP and to incorporate the extracted sequence into PUC18 vector since this vector is the one used to express full PLP (scheme 23). Also, it was important to maintain the His6 tag and the TEVc/site for purification on Ni-NTA column. For this, we proceeded by amplifying two fragments of the full PLP expression vector: One fragment contained the PLP's active site and the second fragment contained the pUC18 vector, the His6 tag and the TEVc/site. The two PCR products were then purified and digested by Dpn1. The two fragments were then Chapter 5: Half proteins assembled using the Gibson assembly method as described in chapter 4.

Transformation and selection resulted in colonies of bacteria that contained a plasmid formed from both fragments as demonstrated by specific PCR amplifications. To confirm the DNA sequence of the plasmid, and to examine the junctions between the fragments, sequencing was done. One selected colony was grown then plasmids were extracted, purified and sequenced. The expected sequence was obtained.

Expression and purification of the His6 -Half PLP

To obtain substantial quantities of protein, the resulting plasmid was used to transform E. coli BL21(DE3) cells. As for the half PMI protein, a pre-inoculum from a single colony was grown in a starter LB-ampicillin culture. TB supplemented with ampicillin was inoculated from the pre-inoculum at 1/100 and grown at 37 °C until the OD at 600 nm reached a value between 0.6 and 0.8. 0.1 mM IPTG was then added and incubation was continued at 20 °C for 18 h. Bacterial cells were pelleted and kept at -20 °C. The bacterial pellets were resuspended in the presence of benzonase and lysozyme. The mixture was then sonicated and centrifuged (RCF=13000) to eliminate all solids. The clear solution containing the His6 tagged protein was purified on an Ni-NTA agarose column. The various fractions eluted from the column were analyzed by SDS-PAGE followed by Coomassie blue staining. No bands were observed in the elution fractions (Data not shown). This can be due either to the low expression or to its expression in the inclusion bodies as it was observed in the case of full His6-PLP (see chapter 4). However, western blot using an anti His6-tag antibody (Figure 94) showed a band with a good intensity corresponding to the protein with a molecular weight of about 11 kDa for the elution fractions. This confirmed that the protein was indeed His6 tagged.

CONCLUSION:

As we mentioned before, PLP and PMI are similar bicupins characterized by a structure formed of two beta barrel domains, each possessing one active site in one of their beta barrel domains.

Aiming at evaluating the maintenance of the initial activity of the protein in the single barrel of β sheets containing the active site without the presence of the second barrel of β sheets, we proceeded to the preparation of each half of the 2 starting enzymes (Half PMI and Half PLP) made of one barrel of β sheets which contains the metal in their active site.

The expression vectors containing the DNA encoding for the Half proteins were successfully constructed and the proteins were expressed in E.coli BL21. Western blot confirmed that both half proteins were indeed expressed but initial purifications were not satisfying. This can be due to folding problems for both Half proteins. As for Half PLP, it can be due to solubility problems deriving from the staring full PLP which was expressed in both supernatant and inclusion bodies (see chapter 5). However, even if the Half proteins are misfolded, the chimeric PMI-PLP could be well folded as it contains two cupin domains which might be necessary for the fold. This result highlights the advantage of developing chimeric proteins.

The optimization of the purifications should thereafter proceed by first checking the solubility of the half enzymes. For this, the produced half proteins will be analyzed on SDS PAGE gel electrophoresis before and after the cell lysis to verify their expression quantity and location.

CHAPTER 6: GENERAL CONCLUSION AND FUTURE WORK

Biocatalysis using natural enzymes is mostly limited to natural substrates of the employed enzyme and to biotic reactions. Starting from synthetic substrates, artificial enzymes that are developed to catalyze chemist-invented reactions are successfully leading to pure enantiomers of industrially relevant products. [START_REF] Matsuo | Artificial enzymes with protein scaffolds: Structural design and modification[END_REF] Metal complexes are the most efficient man-made catalysts and are also routinely used to form the catalytic cofactors of artificial metalloenzymes. The latter combine both the advantages of natural metalloenzymes such as stereoselective catalysis in aqueous solution, under mild conditions and the advantages of chemically synthesized metal complexes catalysts such as chemist-invented catalysis and compatibility with artificial substrates. [START_REF] Ghattas | Receptor-Based Artificial Metalloenzymes on Living Human Cells[END_REF] Different strategies were described over the years to construct artificial metalloenzymes. But in this project, a new strategy seemed most appealing to us:

Development of chimeric metalloenzymes. We prepared an artificial chimeric protein that possesses multiple active sites by the fusion of catalytic domains of two related enzymes. This chimeric enzyme would be used to catalyze reactions in cascade under ecofriendly conditions.

To prepare the chimeric enzyme, we relied on the cupin superfamily of proteins.

Therefore, the chimeric enzyme combines the catalytic domains of two similar bicupin proteins: PMI and PLP, which should avoid denaturation and lead to a correct folding.

Prior to the preparation of the chimeric PMI-PLP metalloproteins we proceeded to express and purify each of the two starting enzymes i.e. PMI and PLP and to test their activities before and after exchanging the metal cations of their respective active site.

We plan to compare these activities with those of the chimeric PMI-PLP metalloproteins.

PMI are metalloenzymes that catalyze the reversible isomerization of D-mannose-6phosphate into D-fructose-6-phosphate their active site is located in a characteristic barrel of β-sheets. [START_REF] Ahmad | Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-D -arabinonhydrazide[END_REF] Artificial enzymes formed by incorporating transition metals or their complexes in β-sheets are rare. Aiming at elucidating the role of such active site, we prepared and characterized an artificial metalloprotein based on PMI. The enzyme expression vector yielded PMI comprising a His6 tag that we were able to cleave by introducing a TEV protease site using molecular biology techniques. The expression plasmid was digested and a site for specific hydrolysis by TEV protease was successfully introduced flanked by the sequence of the enzyme and that of the His6 tag. After Chapter 6: General Conclusion and Future work purification on a Ni-NTA column, the enzyme was separated from the His6 tag by digestion with TEV protease. TYCHO showed that the thermal denaturation of PMI occurred at 60°C. PMI contains one zinc(II) per enzyme molecule and the removal of zinc(II) from the active site was performed by incubation with 10 6 equiv. EDTA. To prepare the artificial metalloenzyme, copper(II) was added and PMI was successfully transformed into an artificial copper(II)-containing protein that was characterized by a relatively good dissociation constant (KD = 3 µM). The EPR spectrum of the PMI-Cu complex confirmed the presence of a single copper in the binding site of the enzyme.

The PMI-Cu complex did not catalyze Diels-Alder reaction most probably because the active site was buried within the enzyme and not accessible for the hallmark substrates of artificial metallo-Diels-Alderases. As perspectives, smaller Diels-Alder substrates will be used with this protein as catalyst. A peroxidase activity is also envisioned for the artificial PMI-Cu protein.

PLP is a metalloenzyme that resembles PMI. It belongs to the cupin superfamily of enzymes, and its active site is located in the characteristic barrel of β-sheets. The biological role of PLP remains unclear. A previous study has shown that PLP could be transformed into an artificial metalloenzyme by substituting its iron cation by a divalent metal cations such as Cu 2+ . [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] Aiming at the preparation of a chimeric PMI-PLP protein, we first prepared and characterized an artificial metalloprotein based on PLP. The PCR products were then purified, annealed and ligated via the Gibson Assembly method. The constructed expression vector containing the DNA encoding for the PMI-PLP chimera was used for expression in E.coli BL21. Initial purification was not successful as the chimera remained contaminated. Since molecular modelling using MODELLER revealed a possible clash between an  helix in the variable region deriving from PMI and the cupin barrel deriving from PLP, a second chimeric enzyme, Chimera 2, was constructed by replacing the PMI loop responsible for the clash by the analog PLP loop which is much smaller. Chimera 2 was also expressed but similarly to chimera 1 the purification here failed again. Indeed, the proteins were found mainly in the insoluble fractions, most probably in inclusion bodies. The co-expression of chaperones proteins with chimera 2 failed aiding solubilization.

Aiming at evaluating the conservation of the initial activity of a bicupin by a single cupin domain containing the active site thus in the absence of the second cupin domain, we proceeded to the preparation of each half of the two starting enzymes (Half PMI and Half PLP) made of one barrel of β sheets which contain the native metal in their active site. Hence, the expression vectors containing the DNA encoding for the Half proteins were constructed and the proteins were expressed in E.coli BL21. Western blot confirmed that both half proteins were indeed expressed but initial purifications Chapter 6: General Conclusion and Future work were also not satisfactory. We suspect that these proteins were also produced in inclusion bodies, which remains to be verified by analysis on SDS PAGE gel electrophoresis before and after the cell lysis. This can be due to folding problems for both Half proteins since they are formed of only one cupin domain. As for Half PLP, it can be due to solubility problems deriving from the staring full PLP which was expressed in both supernatant and inclusion bodies.

For Chimeric proteins, first, alternative methods of purification such as the use of denaturing agents and solubilization should be examined. Hence, the protein should be denatured which will require its subsequent refolding. Second, another possible solution leading to correctly folded chimera could be to reverse the order of the cupin domains. As mentioned earlier, the order of insertion influences the folding. [START_REF] Yu | Synthetic fusion protein design and applications[END_REF] Inspired by natural and artificial enzymatic catalysis, the aim for preparing the chimeric PMI-PLP enzyme is the catalysis of reactions in cascade. Obtaining a correctly folded chimeric bicupin enzyme remains difficult, then mutating the metal-free domain of a bi-cupin is another option to achieve our aim. Indeed, inspired by the metal binding cupin domain of PLP, site directed mutagenesis may be used to create a metal binding site in the metal free cupin domain of PMI for example. This less challenging strategy could nevertheless be more successful to reach our goals. In fact, inspired by the different metal binding site of cupins, site directed mutagenesis can be used to adapt a binding site for a specific metal cation for a sought after activity. This could facilitate the preparation of enzymes with different activities while maintaining the same core of the bicupin domains.

Once such protein obtained, a starting point for analyzing catalysis will be to assay the isomerase activity deriving from the parent PMI so that the impact of the Fe(III) site on this reaction can be identified (scheme 24). Finally, the reconstitution of the artificial enzyme by a different metal in each active site may be achieved by removing only the Fe(III) cation using a ligand that possesses high affinity for Fe(III) but low affinity for Zn(II) such as deferasirox (a drug that is 20 times more selective for Fe(III) than for Zn(II)). The reconstitution of the enzyme by a Cu(II) containing active site should provide the quercetinase activity while retaining the Zn(II) containing active site and its isomerase activity. These two very different active sites can then be used to catalyze different reactions, as well as reactions in cascade such as quercetin oxidation catalyzed by the artificial copper site of PMI, followed by the hydrolysis of the product catalyzed by the zinc of PMI (scheme 26).

MATERIALS AND METHODS

I. CONSTRUCTION OF THE EXPRESSION VECTOR OF THE PROTEINS:

-

Plasmid purification:

o For PMI :

BL21 (DE3) E. coli cells incorporating a version of the pRSET-C expression vector (total 8 kb) containing the expression system of the His6 tag PMI and the ampicillin resistance gene (see annex 1) were purchased from Sigma. The bacterial strain was first thawed and then grown at 37°C for 18 hours on plates coated with LB-agar (Lysogeny Brothagar, Sigma) medium supplemented with ampicillin (100 μg/mL, Sigma).

A pre-inoculum from a single colony was grown in a 3 mL starter LB-Ampicillin culture at 37 °C (100 μg/mL). When the OD at 600nm (Cary 50, Varian) reached a value around 4 (after 6 hours), LB supplemented with 100 μg/mL ampicillin was inoculated with the preinocculum (final dilution 1/1000 th ) and the cells were grown at 37°C overnight (225 rpm).

The plasmid DNA was then purified by using the Xtra MIDI Kit (MACHEREY-NAGEL).

The culture was centrifuged at 6000 g for 15 minutes, the supernatant was then discarded and the pellet was resuspended in resuspension RES buffer. Lysis buffer (sodium hydroxide<2%, sodium dodecylsulfate) was added to the suspension and incubated for 5 minutes at room temperature after inverting the tube 5 times.

Neutralization Buffer NEU (acetic buffer) was added to the suspension, the lysate was mixed gently by inverting the tube until the blue colored samples turned completely colorless, and it was then incubated on ice for 5 minutes. The NucleoBond® Xtra

Column was equilibrated as recommended, the lysate was then loaded onto the column, washed 3 times using the washing buffer (diluted ethanol <20%, potassium o For PLP :

The plasmid which provided ampicillin resistance and the expression vector for the His6-PLP was requested from Kataoka's team in Japan (see annex 2). [START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] The lyophilized plasmid resuspended in sterile water was used to transform E. coli (DH5alfa) strain (Sigma). The cells were transferred from -70°C to wet ice and thawed for 10 minutes. The DNA was diluted to 10 ng/μL in water and then added directly to 100 μL competent cells in 14-ml BD Falcon polypropylene round bottom tube, that were mixed by gentle tapping, and incubated on ice for 30 minutes. The cells were transferred to a 42° C water bath for exactly 45 seconds and then back to ice for 2 minutes. 450 µl of SOC medium (Sigma) was added and the cells were incubated on a shaker incubator (225 rpm) at 37°C for 1 hour. Transformed cells were suspended into LB agar plates with ampicillin (75 μg/mL) and were incubated at 37°C overnight (225 rpm). A pre-inoculum from a single colony was grown in a 5 mL starter culture in LB-Ampicillin at 37 °C (75 μg/mL).

The plasmid DNA was then purified by using NucleoSpin Plasmid transfection-grade (MACHEREY-NAGEL): The culture was centrifuged at 11000 g for 1 minute, the supernatant was discarded, and the pellet was resuspended in Resuspension A1 buffer.

Lysis buffer was added to the suspension and incubated for 5 minutes at room temperature by inverting the tube 5 times. Neutralization Buffer was added to the suspension, the lysate was mixed gently by inverting the tube until the blue colored samples turned completely colorless, and it was then centrifuged 10 minutes at 13000g.

The clear supernatant was then loaded onto the given kit column and washed 3 times.

The DNA was then eluted by adding 25 μL of sterile water. To inactivate the enzyme, the next day, the mixture was heated at 65°C for 25 minutes.

o For Chimera 2 :

The pET-28b vector and the synthetic DNA sequence encoding for the chimera 2 were double digested with BamH1 (New England Biolab: NEB) and Pst1 (NEB). For this, 1μg

DNA was added to 1μL BamH1, 1 μL Pst1, 5 μL Cutsmart buffer (50 mM Potassium Acetate. 10 mM Magnesium Acetate. 100 µg/ml BSA. pH 7.9) (NEB) and 33 μL sterile water. The mixtures were then incubated 4 hours at 37°C.

-

Agarose gel electrophoresis and ethidium bromide staining:

The 1% agarose gel was prepared by dissolving agarose powder (1 g, Sigma) in 1x Tris Borate EDTA (TBE) buffer, pH = 8 (100 mL, Sigma) supplemented with ETBr (0.2-0.5 μg/mL, Sigma) and a comb was placed to create wells for loading samples. Once the gel was set, the comb was removed, leaving wells wherein DNA molecular weight ladder and samples of circular plasmid (3.2 μg/μL) and of the digesting mixture (1.2 μg/μL) were loaded mixed with low molecular weight dye (sigma).

Nhe1

After 30 minutes of migration at a voltage of 100 Volts, the dye reached the edge of the gel. The electric current was turned off and the gel was visualized with an ultraviolet (UV) DNA transilluminator (Biorad).

For PMI, a DNA band of 8 kb corresponding to the digested plasmid was cutout and purified.

For the construction of the chimera 2, a DNA band of 6379 pb corresponding to the digested pET-28B plasmid was cutout and purified. In addition, a DNA band of 1330 pb corresponding to the digested sequence of the Chimera 2 was also cutout and purified.

-

DNA extraction from agarose gels:

The extraction from agarose gel was performed using the MACHERY-NAGEL kit (PCR clean up and gel extraction). 100 mg of gel containing the linear digested plasmid were added to 200 μL buffer NT1 (guanidine thiocyanate) and the sample was vortexed briefly until the agarose gel was completely dissolved (5-10 min at 50°C). The sample was loaded onto the provided NucleoBond® Xtra Column and centrifuged for 30 s at 11000 g. Buffer NT3 was then added, the sample centrifuged for 30 s at 11000 g. The flow through was discarded. The DNA was then eluted from the column by adding Buffer NE, incubated at room temperature (18-25 °C) for 1 min and then centrifuged for 1 min at 11000 g.

For the PMI, the linear plasmid concentration (33 ng/µl) and purity were also measured using the nanodrop spectrophotometer (Thermo Scientific) apparatus (A260 = 0.669; A280 = 0.366; A260/A280 = 1.83).

For the Chimera 2, the linear digested plasmid was eluted by adding 20 μL of sterile water. The DNA concentration (24 ng/μL) was determined by measuring the UV-vis absorption spectra of samples on a nanodrop spectrophotometer apparatus (ThermoFisher). On the other hand, 29 ng/μL DNA encoding for the synthetic sequence of the chimera 2 was eluted.

-

Modification of the plasmid:

o For PMI:

12 µl of the purified vector (33 ng/µl) was mixed with 4 µl of DNA insert (10 molar equivalents), 2 µl 10x T4 DNA ligase buffer (500 mM Tris, 100 mM MgCl2, 50 mM DTT, 10 mM ATP, pH=7.6, Sigma), 2 µl T4 ligase (1U/μL, Sigma), following the providers (Sigma) procedure. The mixture was then incubated overnight at 8°C.

The ligation mixture was used to transform E. coli (DH5alfa) strain (Sigma). The cells were transferred from -70 °C to wet ice and thawed for 10 minutes. 50 μL of the competent cells were aliquoted into 14-ml BD falcon polypropylene round bottom tube. 5 μL of the ligation mixture was added directly to competent cells, mixed by o For chimera 2:

2.5 µl of the purified vector (24 ng/µl) was mixed with 2.5 µl of DNA insert (29 ng/µl) and 5 µl instant sticky ligase (NEB), following the providers (NEB) procedure. The mixture was then incubated 15 minutes in an ice bath.

The ligation mixture was added directly to 50 µl competent cells in 14-ml BD Falcon The presence of the PCR amplicon and the size of the product were determined by electrophoresis alongside a DNA size marker on a precast 1.2% agarose gel.

o For PLP:

-Deletion of a small region of the lacZ promoter:

25μL of the PCR mixture was prepared using the Q5 site directed mutagenesis kit by adding 12.5 μL 2x Master Mix Q5 hot start kit (NEB) containing the polymerase and its buffer, 2.5 μL primers (final concentration 10 µM), 9 μL nuclease free water and 1 μL of the plasmid (10 pg -1 μg).

The PCR was carried out using two primers (P1: Then, the PCR product was treated by Kinase, Ligase and DPN1 enzymes (KLD). For this, 1 μL of the PCR product was mixed with 1 μL KLD enzyme mix 10x, 5 μL 1X KLD reaction buffer 2x and 3 μL nuclease free water.

-TEV Protease site's insertion:

The TEV protease site (5' GAA AAC CTG TAT TTT CAG GGC 3') was inserted by PCR mutagenesis using two primers (P1: 5' TTT TCA GGG TCA TCA TCA TCA TCA TCA TTG AGG ATC CT 3' and P2: 5' TAC AGG TTT TCG GCC AGT GTC CCA TCG CGA AAA TC 3').

The PCR mixture was prepared using the Q5 site directed mutagenesis kit by adding 12.5 μL 2x Master Mix Q5 hot start kit (NEB) containing the polymerase and its buffer, 2.5 μL primers (final concentration 10 µM), 9 μL nuclease free water and 1 μL of the plasmid (10 pg -1 μg). The PCR was carried out in an applied biosystem thermocycler (StepOne Plus). It started with 30 seconds at 98°C for Taq polymerase activation Materials and methods followed by 25 4,5-minutes cycles including 10 seconds at 98°C for DNA denaturation, 20 seconds at 60°C for hybridation, 2 minutes at 72°C for extension and 2 minutes at 72 °C for final extension.

The presence of the PCR amplicon and the size of the product were determined by electrophoresis alongside a DNA size marker on a precast 1.2% agarose gel.

o For Chimera 1:

The pRSET-C vector for we previously used for expressing PMI was chosen as a vector (see annex 3). The suitable sequences of PMI and PLP were amplified. The PCR mixtures were prepared using the Q5 site directed mutagenesis kit by adding 12.5 μL 2x Master Mix Q5 hot start kit (NEB) containing the polymerase and its buffer, 2.5 μL primers (final concentration 10 µM), 9 μL nuclease free water and 1 μL of the plasmid (10 pg -1 μg).

Each PCR was carried out in an applied biosystem thermocycler (StepOne Plus) using PCR products were purified and treated with Dpn1(Thermofisher, 10 u/ µL). 1 µg of eluted DNA in sterile water was mixed with 1µL of Dpn1 solution, 2 µL of 2x Tango buffer, the solution was completed to 20 µL with nuclease free water. The mixture was incubated 16 h at 37 °C after which the enzyme was inactivated at 80°C for 20 min.

Then, the digested PCR products (1 µl PMI and 3 µl PLP) were assembled using the NEBuilder Hifi DNA assembly reaction kit by adding 10 µl Hifi DNA assembly Master Mix and 6 µl nuclease free water for 20 minutes at 50°C before transformation.

o For Chimera 2:

The synthetic sequence encoding for the chimera 2 was amplified using two primers: Materials and methods in ice for 30 minutes. The cells were then transferred into a 42° C water bath for exactly 45 seconds and then back into ice for 2 minutes. 650 µl of SOC medium (NEB) were added and the mixture were incubated in a shaker incubator (225 rpm) at 37°C for 1 hour. 100 μl transformed cells were coated on LB agar plates with ampicillin (100 μg/mL) and the plates were incubated at 37°C overnight (225 rpm).

o For Half PLP:

The sequence was amplified using the Q5 HiFi PCR NEB kit. The DNA sequence of Half PLP was amplified using four primers:

-P1: 5' AGC TAT GGG CCG GCC TAC ATC CGA TG 3' and P2: 5' CAG GTT TTC TGC GAA GTC GCG GTA ACC CG 3' for the sequence encoding for the PLP's active site.

-P3: 5' ACT TCG CAG AAA ACC TGT ATT TTC AGA GTC ATC ATC ATC ATC ACA TTG AGG 3' and P4: 5' GGC CGG CCC ATA GCT GTT TCC TGT GTG AAA TTG AAA TTG TTA TCC GC 3' for the sequence encoding for the pUC18 vector.

Each PCR was carried out in an applied biosystem thermocycler (StepOne Plus). It started with 30 seconds at 98°C for Taq polymerase activation, followed:

-For PMI, by 25 5-minutes and 10-seconds cycles, including 10 seconds at 98°C for denaturation, 20 seconds at 66°C (PMI's active site) and 20 seconds at 64°C (pUC18 vector) for hybridization, 140 s at 72°C for extension and two minutes at 72 °C for primer final extension.

-For PLP by 25 3-minutes and 5-seconds cycles including 10 seconds at 98°C for denaturation, 20 seconds at 66°C (PLP's active site) and 20 seconds at 64°C (pUC vector) for hybridization, 15 s at 72°C for extension and two minutes at 72 °C for primer final extension.

PCR products were purified using the Macherey-Nagel kit. Concentration and purity were assayed on nanodrop spectrophotometer (Thermo Scientific) and treated with Dpn1 enzyme (Thermofisher, 10 U/µL). 1 µg of DNA was mixed with 1µL of Dpn1 solution, 2 µL of 2x Tango buffer, the solution was completed to 20 µL with nuclease free water. The mixture was incubated 16 h at 37 °C and then the enzyme was inactivated at 80°C for 20 min. Then, the digested PCR products were assembled using the (NEB) HiFi DNA assembly reaction kit by adding 10 µl Hifi DNA assembly Master Mix and 6 µl nuclease free water for 20 minutes at 50°C before transformation. Materials and methods

-

Sequencing:

For PMI, the sequencing was performed on purified plasmids at Eurofins using the Sanger method, in a single direction, starting from the expression T7 promotor. One direction sequencing was sufficient to confirm the PCR results since the remainder of the protein sequence was not modified. The single direction sequencing of PLP was performed following the same procedure, starting from the expression M13 Forward, which was sufficient to confirm the PCR results. The Sequencing of the chimeric enzymes was also performed on purified plasmids at Eurofins, using the Sanger method but in two directions using either T7 promotor or T7 Term.

-

Transformation of bacteria:

To obtain large quantities of protein, the modified plasmid containing the DNA encoding for PLP was used to transform E. coli BL21(DE3) strain (Sigma-Aldrich), while for the modified plasmid containing the DNA encoding for PMI, E. coli BL21(DE3) pLysS strain was used. The transformation was performed according to the same procedure as that described above.

II. EXPRESSION AND PURIFICATION OF THE PROTEINS:

-

TEV protease

Tobacco Etch Virus (TEV) proteases recognizes the seven-amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly that it cleaves between Gln and Gly with high specificity. In this project, two TEV proteases have been used: His6 tagged MBP-TEV protease that we have purified in our laboratory following the protocol described in the literature 108 and AcTEV Protease (His6-AcTEV) which is commercial and can be purchased from Sigma. AcTEV Protease, an enhanced form of (TEV) protease, is highly site-specific and active, and it is more stable than native TEV protease.

First, the pYFJ16 plasmid which provided ampicillin resistance and the expression vector for the His6 tagged MBP-TEV protease (His6-Maltose Binding Protein (MBP)-TEV) was obtained in an E. coli DH5 alpha bacterial stab from Addgene. A subculturing was made on plates coated with LB medium supplemented with 75 µg/mL ampicillin and cells were grown overnight. Then, the plasmid was extracted using the MACHEREY-NAGEL kit as described above and its sequence was verified by sequencing. E. coli BL21codon plus competent cells were then transformed with the sequenced plasmid and were grown overnight in 1L TB medium supplemented with 75 µg/ml ampicillin at 37°C, with shaking (200 rpm). The expression of the recombinant protein was induced with 0.1 mM IPTG. The purification of His6-MBP-TEV was performed on a Ni-NTA affinity column following the protocol described below. Starting from a 800 mL culture, 60 mL of 70 µM TEV protease were obtained.

-

Protein purification:

For PMI and PLP, a pre-inoculum from a single colony was grown in a starter culture in 20 mL LB supplemented with ampicillin (75 μg/mL), at 37 °C for 18 hours. 2L TB (Terrific Broth) supplemented with ampicillin (35 μg/mL) was inoculated with the preinocculum (1/100 th dilution) and grown at 37 °C until the OD600 reached a value between 0.6 and 0.8 (after 3 hours). At this stage, a final concentration of 0.6 mM isopropyl β-D-1thiogalactopyranoside (IPTG) was added and incubation was continued at 20 °C for 18 h. Bacterial cells were pelleted by centrifugation and resuspended in lysis buffer (50mL Lysis buffer for 1L bacterial culture) (50 mM Tris HCl, 300 mM NaCl, 10% Glycerol, 1%

Triton, 5 mM imidazole, pH = 8). After adding Lysozyme (10 mg/mL) and benzonase (3 μL benzonase for 30 mL lysis buffer) the sample became viscous. The sample was sonicated on ice (Amplitude = 50 %, 12 minutes, 15 s ON, 30 s OFF) and then centrifuged at 13000 g for 45 minutes at 4°C. The supernatant was collected for purification by column chromatography on an AKTA purification system. The supernatant was applied onto a Ni-NTA column (HisTrap™ HP GE Healthcare Life Science; column volume, 5 mL), which had been equilibrated with buffer A (flow rate, 5 mL min-1) using an ÄKTA prime plus (GE Healthcare). The column was washed with 100 ml of buffer A at a flow rate of 5 mL min -1 . Elution of the appropriate protein was achieved using a linear gradient 50% of buffer A and 50% of B 0% at a flow rate of 2 mL min -1 (Buffer A: 75 mM Tris hydrochloride pH 7.5, 300 mM NaCl, and 20 mM imidazole; buffer B: 5 mM Tris hydrochloride pH 7.5, 300 mM NaCl, and 250 mM imidazole).

Starting from 1500 mL of culture, 40 ml of pure His tagged proteins (1g/L) were obtained.

The purity of the proteins was verified by SDS-PAGE analysis followed by Coomassie blue staining (see below). The eluted PMI was dialyzed against a 50 mM MOPS buffer, pH=7.4; whereas, the eluted PLP was dialyzed against a 50 mM MOPS buffer, 300 mM NaCl, pH = 7.6. His tagged proteins were then concentrated on vivaspin20 10 kDa cut off.

The concentrations were obtained from the absorbance at 280 nm measured on a Nanodrop spectrophotometer (Thermo Scientific) using extinction coefficients at 280 nm calculated from the protein sequence (https://web.expasy.org/protparam/) (28 880 M -1 .cm -1 for His6-PMI, 21 430 M -1 .cm -1 for His6-PLP, 75 190 M -1 .cm -1 for His6-MBP-TEV protease and 39 880 M -1 .cm -1 for His6-Chimera 1).

-

Cleavage of the His6 tag:

o By Active Tobacco Etch Virus (AcTEV) protease:

In order to cleave the His6 tag, 1 mL of the purified and concentrated protein (3.48 mg/mL) was mixed with 20 μL of AcTEV protease (10 U/µl, ThermoFisher), 135 μL of 20x Buffer and 10 μL of 0.1M DTT and the resulting solution was incubated at 4°C overnight.

Following digestion, the AcTEV protease terminus was removed from the cleavage reaction mixture by purification on a Ni-NTA affinity column as described above. Pure tag free protein could not bind on the Ni-NTA affinity column and was thus recovered in the flow through. It was dialyzed against 50 mM MOPS buffer, pH=7.1 and concentrated via vivaspin20 cut off.

o By His6 tagged MBP TEV protease (His6-MBP-uTEV3):

His-MBP-uTEV3 was first purified in our lab following the described protocol. 108 In order to cleave the His6 tag, 35 mL of the purified and concentrated protein (28 μM in 50mM MOPS buffer) was mixed with 2,8 mL TEV protease (70 μM) and 760 μL of 0.1M DTT (Cf DTT=2 mM) and the resulting solution was incubated at 4°C overnight.

Following digestion, AcTEV protease terminus was removed from the cleavage reaction mixture by purification on a Ni-NTA affinity column. The purification was performed according to the same procedure as that described above. Pure tag free protein could not bind on the Ni-NTA affinity column and thus was recovered in the flow through.

The concentrations of the proteins were obtained from the absorbance at 280 nm measured on a Nanodrop spectrophotometer (Thermo Scientific) using extinction coefficients at 280 nm of 27 390 M -1 .cm -1 for PMI and 20 220 M -1 .cm -1 for PLP as calculated from the protein sequence (https://web.expasy.org/protparam/).

-

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining:

The The bands could then be revealed by adding the chemiluminescent substrate provided by Thermo Scientific containing solutions of hydrogen peroxide and luminol which generated light that could be detected on an Image Quant LAS 500 apparatus.

III. CHARACTERIZATION OF THE PROTEINS:

-

Enzyme activity assay of PMI

To assay the catalytic activity of PMI for the isomerisation of M6P into F6P, a test involving three steps and two other enzymes PGI (phosphoglucose isomerase) and G6PDH (glucose-6-phosphate dehydrogenase) was set up in the lab (Scheme 7.1). [START_REF] Ahmad | Crystal structure of phosphomannose isomerase from Candida albicans complexed with 5-phospho-D -arabinonhydrazide[END_REF] PGI first transforms F6P formed by PMI into G6P, which is further transformed into 6PGL (6-phosphogluconolactone) by G6PDH. The later reaction produces NADPH that can be detected by measuring the absorbance at 340 nm ( = 6220 M -1 .cm -1 , see scheme 28). PGI and G6PDH are introduced in large excess (100 equiv.) with respect to PMI, so that the rate determining step for forming NADPH becomes the reaction catalyzed by PMI. The amount of NADPH formed is thus proportional to the amount of M6P isomerize by PMI.

Scheme 28: Three reaction system for measuring PMI activity.

PGI (from rabbit muscle) and G6PDH (from yeast) were commercial enzymes provided by Sigma Aldrich. For PGI, the commercial solution was stored at 4 ⁰C. 500 μL of distilled water were added to the commercial enzyme solution. This solution was aliquoted by 15 μL. The aliquots were stored in the freezer at -20 ⁰C. From these aliquots, solutions PGI/100 were then prepared with 50 mM HEPES buffer pH 7.4. For G6PDH, the commercial solution was stored at 4 ⁰C. 500 μL of distilled water were added to the commercial enzyme solution. This solution was aliquoted by 15 μL. The aliquots were stored in the freezer at -20 ⁰C. From these aliquots, solutions G6PDH/100 were then prepared with 50 mM HEPES buffer pH 7.4. The PGI/100 and the G6PDH/100 solutions were used for the PMI activity assay.

All the substrates and enzymes except PMI (Table 4) were first added in a 1 cm quartz cell for UV-visible spectrocopy. After 7 minutes of incubation at 25°C, increasing volumes of 12 M PMI (30-120 L) were added and the resulting solution was mixed by pipetting up and down. The kinetics was followed by measuring the absorbance at 340 nm (A340) every ten seconds for 10 minutes. The initial rate of the reaction catalyzed by PMI could be determined from the slope (s) at the origin of the curve A340 = f(t) :

Vi(mM/min) = s/6.22). Vi was measured at various PMI volumes (V) and the slope of the Vi = f(V) provided the specific activity of the per µL of protein U/µL. The removal of zinc from the active site of the PMI was performed by incubating 10 mL of a solution of the protein (3 µM in 50 mM MOPS buffer, pH = 7.4) with 10 6 equiv.

EDTA (Ethylenediamine-tetraacetic acid) overnight at 4°C with shaking. Then, the protein was washed 3 times through a vivaspin20 10 kDa cut off by 30 mL of a 50 mM MOPS buffer pH 7.4 and was finally dialyzed against 2 L of a 50 mM MOPS buffer, pH 7.4 for 2 days. Ces deux sites actifs très différents peuvent alors être utilisés pour catalyser différentes réactions, ainsi que des réactions en cascade comme l'oxydation de la quercétine catalysée par le cuivre du site actif de la PLP, suivie par l'hydrolyse du produit ainsi formé catalysée par le zinc de la PMI.

LIST OF FIGURES:

-----

  Introduction ............................................................................................................................................. A. General context......................................................................................................................... A.1. Searching for new bio-compatible biocatalysts ....................................................... A.2. Chimeric enzymes ................................................................................................................ B. Aim of the project .................................................................................................................... B.1. Starting enzymes: PMI & PLP........................................................................................... B.2. The strategy to produce the Chimeric enzyme ......................................................... Chapter 1: State of the art .................................................................................................................. 1.1. Artificial metalloenzymes.................................................................................................. 1.1.1. Different strategies to construct artificial metalloenzymes: .......................... Covalent attachment ....................................................................................................... Supramolecular anchoring ............................................................................................ Metal exchange ................................................................................................................. -Directed evolution ............................................................................................................ -Fusion of multiple proteins ........................................................................................... 1.1.2. Some examples of reactions catalyzed by artificial metalloenzymes ........ 1.2. Chimeric enzymes ............................................................................................................... 1.2.1. Fundamental types of chimeric proteins: Type 1 & Type 2 .......................... 1.2.2. Choice of the linker ...................................................................................................... 1.2.3. Design of chimeric enzymes ..................................................................................... 1.2.3.1. Structure's prediction:......................................................................................... 1.2.3.2. Construction of fusion proteins ...................................................................... Tandem fusion ............................................................................................................... -Domain insertion .......................................................................................................... -Post translational protein conjugation: ................................................................ Table of contents 1.2.4. Applications of the fusion proteins: ....................................................................... 1.2.4.1. Multifunctional biocatalytic activities:........................................................... 1.2.4.2. Biosensing and bioactivity regulation:.......................................................... 1.2.4.3. Creating peptide libraries .................................................................................. 1.2.4.4. Bio-therapeutics applications .......................................................................... -Immunotechnology ..................................................................................................... -Creating new drugs with targeted delivery to tumor cells ............................ Humanized antibodies ................................................................................................ -Some examples of fusion therapeutic's proteins: ............................................. 1.3. Cupin proteins ...................................................................................................................... 1.4. Conclusion ............................................................................................................................. Chapter 2: Phosphomannose Isomerase ...................................................................................... 2.1. Introduction: ............................................................................................................................... 2.2. State of the art ...................................................................................................................... 2.2.1. Different types of PMI: classification of Proudfoot .......................................... Table of contents 5.2.1. Half PMI protein ..............................................................................................................122 5.2.1.1. Construction of the expression vector for Half PMI ..................................122 5.2.1.2. Expression and purification of the His6-Half PMI ........................................123 5.2.2. Half PLP protein ...............................................................................................................125 5.2.2.1. Construction of the expression vector for Half PLP ..................................125 5.2.2.2. Expression and purification of the His6 -Half PLP .....................................127 5.3. Conclusion: ..........................................................................................................................128 Chapter 6: General Conclusion and Future work .....................................................................129 Materials and methods ......................................................................................................................134 I. Construction of the expression vectors of the proteins: ..........................................134 -Plasmid purification: ..................................................................................................134 -Plasmid digestion : .....................................................................................................136 -Agarose gel electrophoresis and ethidium bromide staining: ...................136 -DNA extraction from agarose gels: ......................................................................137 -Modification of the plasmid: ..................................................................................137 -Polymerase chain reaction (PCR): .........................................................................138 -Sequencing: ..................................................................................................................143 -Transformation of bacteria: .....................................................................................143 II. Expression and purification of the Proteins: ................................................................144 -TEV protease.................................................................................................................144 -Protein purification: ...................................................................................................144 -Cleavage of the His6 tag: ..........................................................................................145 -Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining: .................................................................................................................................146 -Western Blot: ................................................................................................................146 III. Characterization of the proteins: ......................................................................................147 -Enzyme activity assay of PMI ..................................................................................147 -Zinc removal: ................................................................................................................148 -Iron removal: ................................................................................................................149 -Protein intrinsic fluorescence: ................................................................................149 -EPR ...................................................................................................................................150 -Isothermal Titration Calorimetry: ..........................................................................150

  .....................................................................................................................................159 List of Schemes .....................................................................................................................................166 Annexes ...................................................................................................................................................174 Annex 1 ...............................................................................................................................................174 Annex 2 ...............................................................................................................................................175 Annex 3 ...............................................................................................................................................176 INTRODUCTION A. GENERAL CONTEXT A.1. Searching for new bio-compatible biocatalysts

Figure 2 :. 13 Scheme 2 :

 2132 Figure 2: Predicted structure of PMI-PLP chimeric enzyme. In red: catalytic domain of PMI; in yellow: catalytic domain of PLP.

Figure 3 :

 3 Figure 3: Superposition of PMI and PLP.

Scheme 3 :

 3 Scheme 3: Design of a chimeric PMI-PLP enzyme that would be able to catalyze cascade reactions.

Figure 4 :

 4 Figure 4: Three strategies for site-specific introduction of transition metals or their complexes into in protein hosts (L = ligand; M = transition metal)

Figure 5 :

 5 Figure 5: Production of enzymes with an optimized catalytic activity following the directed evolution strategy.23

Chapter 1 :

 1 Scheme 5: Formation of chimeric gene by deleting 3' translational term of the gene 1 and the 5' translational term of the gene 2.

Figure 6 :

 6 Figure 6: Crystal structure of ACCO proving that it belongs to the cupin superfamily of enzymes and revealing its iron(II) containing active site.

1 :

 1 State of the art tissue that contains cells with different genes than the rest of the person, organ, or

Figure 7 :

 7 Figure 7: Two fundamental types of chimeric proteins: Type 1: The two proteins or protein subunits are united end-to-end and usually linked by a linker; type 2: the amino acids from both donors are interspersed in the fusion protein product. 8

Chapter 1 :

 1 State of the artIn 2013, Meister and Joshi 45 inserted the calmodulin domain (CaM) into Beta lactamase (BLA). They took advantage of the CaM's capacity to change conformation (Figure10).

Figure 10 :

 10 Figure 10: Structures showing the conformational changes of CaM in the closed state (pdb:

28

 28

Figure 11 :

 11 Figure 11: Schematic representation of the recombinant TNF -IL-12 protein

Figure 12 :

 12 Figure 12: Characteristic structure of cupin proteins showing two conserved 2--strands domains separated by a less conserved inter-motif region containing another 2--strands and a loop.

Figure 13 :Figure 14 :

 1314 Figure 13: Crystal structure of ACCO showing its unique cupin domain and revealing its Fe 2+ containing active site.

Figure 15 :

 15 Figure 15: An example of multicupin: Human homogentisate dioxygenase (HGO).

2 .

 2 Scheme 9: Reversible isomerization of D-mannose-6-phosphate (M6P) to D-fructose-6phosphate (F6P) catalyzed by PMI.

Figure 16 :

 16 Figure 16: Schematic representation of the structure of PMI showing a large inter-domain between two conserved barrels containing Zinc in the active site located on the C-terminal side.

Figure 18 :

 18 Figure 18: View of the metal binding site in PMI (pdb : 1PMI). Interatomic distances are given in Å.

Scheme 11 :

 11 Scheme 11: Structure of the HEI analogue inhibitor 5PAHz.

Figure 20 :

 20 Figure 20 : Agarose gel stained with ETBr of the undigested (Lane 3) and digested plasmid (lanes 1 and 2). Lane 3 shows 3 possible plasmid conformations.

Figure 21 :

 21 Figure 21: Ligation of the insert coding for TEVc/site and the vector linearized plasmid. The insert is complementary to the remaining Nhe1 fragments of both vector strands.

Figure 22 :

 22 Figure 22: Fragment of the sequence encoding for His6-PMI and comprising the inserted TEVc/site with the insert specific primer (violet frame, primer mix 1) that was used for bacterial colony-screening.

Figure 23 :

 23 Figure 23: Agarose gel electrophoresis of PCR products obtained using the positive control primers (primer mix 2) which are complementary to the fragments of the sequence encoding for ampicillin resistance. Lane 1: DNA Ladder

Figure 24 :

 24 Figure 24: Agarose gel electrophoresis of PCR products obtained using the primer mix 1 which is complementary to fragments of the sequence of the insert i.e. TEVc/site and the vector. Lane 1: DNA Ladder

Figure 25 :

 25 Figure 25: Coomassie blue stained SDS-PAGE of supernatant fractions and pellets after the expression of the PMI. Lanes 1, 3, 5 and 7: Supernatant fractions; Lanes 2, 4, 6 and 8: Pellet fractions L: Protein Ladder.

Figure 26 :

 26 Figure 26: Coomassie blue stained SDS-PAGE of fractions collected after the purification on an Ni-NTA agarose column of His6-PMI. Lanes 6, 7, 8 ,9, 10, 11 and 12 show bands corresponding to the expected molecular weight of pure His6-PMI (of about 50 kDa); L: Protein Ladder.

Figure 27 :

 27 Figure 27: Coomassie blue stained SDS-PAGE gel of fractions collected after the purification. Lane 1: supernatant; Lane 2: washed fraction; Lanes 3 and 4 show bands with high intensity corresponding to the molecular weight of pure His6-PMI (about 50 kDa); L: Protein Ladder.

Figure 28 :

 28 Figure 28: Left: Coomassie blue stained SDS-PAGE; Right: Western Blot image. In Both cases lanes: 1-PMI His; 2-PMI-His + AcTEV; 3-Flow through; 4-Eluted fractions. L: Protein Ladder

Scheme 12 :

 12 Scheme 12: The three successive reactions used to assay PMI activity by uv-visible spectroscopy. Λmax NADPH = 340 nm

Figure 29 :

 29 Figure 29: Pure His6-PMI and tag free PMI specific activity (AS) as function of the volume of enzymes at 25°C in 50 mM MOPS, pH 7.4 in the presence of all substrates (M6P and NADP + ) and auxiliary enzymes (PGI and G6PDH) as indicated. The reported values are initial velocity (slope of the kinetic divided by the extinction coefficient of NADPH).

Figure 30 :

 30 Figure 30: Percentage of the PMI activity as function of added EDTA equiv.

Figure 31 :

 31 Figure 31: Fluorescence titration of the binding of Cu(NO3)2 to zinc free PMI upon excitation at 280 nm. Increase of the signal with increasing concentrations of added Cu 2+ (0 to 55 equiv.). Measurements were performed in 50 mM MOPS, pH = 7.4 using 4.5 µM PMI.

Figure 32 :

 32 Figure 32: Curve fitting the concentration of Cu-PMI derived from the fluorescence values at 340 nm plotted against total added Cu(II) concentration.

Figure 33 :

 33 Figure 33: X-band EPR spectrum measured at 10 K of A) Cu(II)-His6-PMI and B) Cu(II)-PMI.

Figure 34 :

 34 Figure 34: TYCHO measurements of natural PMI-Zn in purple; PMI zinc free in orange and Cu-PMI metalloprotein in pink.

Scheme 13 :

 13 Scheme 13: Representation of Diels-Alder cycloaddition reaction of CP and Azachalcone by the artificial Cu-PMI enzyme

Figure 35 :

 35 Figure 35: Left: Structure of PMI crystallized with 5PAHz; Inset: zoom on the active site.

Scheme 14 :

 14 Scheme 14: representation of Diels-Alder cycloaddition reaction of Furan and Acrylonitrile by the artificial Cu-PMI enzyme.

Scheme 15 :

 15 Scheme 15: Substrate design's strategy

Scheme 16 :

 16 Scheme 16: Oxidation reaction of ABTS by H2O2.

Figure 36 :

 36 Figure 36: Swiss model of PLP showing its β barrel catalytic domain surrounded by an αhelixes domain on one side and a C-terminal β barrel domain on the other side.

Figure 37 :Figure 38 : 14 Figure 39 :

 37381439 Figure 37: A) X-ray crystal structure of the human Pirin protein (pdb: 1J1L). B) Swiss model of the PLP metalloprotein.

Figure 40 :

 40 Figure 40: Structure's resemblance of PMI and PLP. Left: Crystalline structure of PMI; Right: Swiss model of the PLP.

Figure 41 :

 41 Figure 41 : Western Blot image of the coexpression of the His6-PLP protein and the Nterminal 10 amino acids of LacZ protein fused His6-PLP protein.

Figure 42 :

 42 Figure 42: The vector linearized plasmid encoding for PLP showing two Shine Dalgarno (SD) sequences. LacZa 10 amino acids were fused to the 33 kDa PLP protein.

Figure 43 :

 43 Figure 43: Primer design for deletion of a portion of LacZa. Forward and reverse primers flank the region to be deleted.

Figure 44 :

 44 Figure 44: The vector linearized plasmid of PLP protein after deletion of a portion of LacZa to avoid the co-expression.

Figure 45 :

 45 Figure 45: Westen Blot image of His6-PLP after the deletion using Anti-His antibody.

Figure 46 :

 46 Figure 46: Insertion of the DNA encoding for the TEV protease cleavage site flanked by the His tag and the PLP linearized sequence.

Figure 47 :

 47 Figure 47: Western Blot image confirming the presence of PCR amplicons. Primary antibody: Anti-His6tag antibody; Secondary antibody: Anti-mouse (H+L) antibody labelled with peroxidase.

3. 3 . 2 .

 32 Expression and purification of the His6-PLP E.coli BL21 cells transformed with the modified plasmid encoding for the His6-PLP-TEVc/site protein were cultured in LB medium supplemented with 75 g/ml ampicillin at 37°C overnight with shaking (200 rpm). The production of the recombinant protein was induced with 0.2 mM IPTG as described in the literature.[START_REF] Widiatningrum | A pirin-like protein from Pseudomonas stutzeri and its quercetinase activity[END_REF] Pellets of bacteria were resuspended in presence of lysozyme and benzonase. The mixture was then sonicated and centrifuged to eliminate all solids. The clear solution containing the His6-PLP was purified on Ni-NTA agarose column. SDS-PAGE followed by Coomassie blue staining (see below) revealed a pure protein with a molecular weight of about 33 kDa (Lanes 6,7 and 8).

Figure 48 :

 48 Figure 48: SDS-PAGE electrophoresis followed by Coomassie blue staining of the fractions collected after the purification of His6-PLP on an Ni-NTA agarose column. Lane 2: supernatant; Lane 3: Flow through; Lane 4: washing fraction; Lanes 5,6,7 and 8 show bands corresponding to the expected molecular weight of pure His6-PLP of about 33 kDa; L: Ladder

Figure 49 :

 49 Figure 49: Western Blot image of His6-PLP. Lanes: 1-Protein Ladder; 2-Flow through; 3-Washed fraction; 5-6-7-8-Eluted fractions.

Figure 50 :

 50 Figure 50: Right, Coomasie blue stained SDS-PAGE electrophoresis of PLP upon cleavage of the His6 tag (on the right) followed by Western Blot image using Anti His primary antibody (on the left). L: Ladder; Lane 1: His6-PLP + TEV; Lane 2: Tag free PLP

Figure 51 :

 51 Figure 51: Fluorescence titration of the binding of Cu(II) to PLP with 10 equiv. EDTA upon excitation at 280 nm. No significant quenching of fluorescence was observed suggesting the remaining of iron cation in the active site of PLP.

Figure 52 :

 52 Figure 52: TYCHO measurements of 10 µM native PLP-Fe in black and 10 µM PLP iron free in pink.

Figure 53 :

 53 Figure 53: TYCHO measurements of 10 µM native PLP in orange and 10 µM PLP iron free upon addition of 10 mM EDTA in purple.

Figure 54 :

 54 Figure 54: Titration of the fluorescence of native PLP upon the addition of increasing amounts (0.2-55 equiv.) of Cu(II) (left) and Zn(II) (right) (excitation at 280 nm). A nonsignificant quenching of the signal was seen in both cases. Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP tag free.

Figure 55 :

 55 Figure 55: Left: Titration of the fluorescence of native PLP upon the addition of increasing amounts of Ni 2+ (excitation at 280 nm). Right: Fluorescence of PLP at 320 nm as function of increasing equivalents of added Ni 2+ . Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP.

Figure 56 :

 56 Figure 56: Quenching of the fluorescence of iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Cu(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP.

Figure 57 :

 57 Figure 57: Curve showing the increase of Cu-PLP concentration, derived from the fluorescence values at 340 nm, as a function of the total concentration of added Cu(II).

Figure 58 :

 58 Figure 58: Quenching of the fluorescence of native iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Zn(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 1.25 µM PLP.

Figure 59 :

 59 Figure 59: Quenching of the fluorescence of native iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Ni(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 1.25 µM PLP.

Figure 60 :

 60 Figure 60: Fluorescence of PLP at 320 nm as function of increasing equivalents of metal cations added. PLP-Cu(II) complex in blue, PLP-Ni(II) complex in orange and PLP-Zn(II) complex in gray.

Figure 61 :

 61 Figure 61: A) concentration of PLP-Ni (derived from the fluorescence values at 340 nm) plotted against the total added Ni(II) concentration. The fit was achieved with n=1. B) total concentration of different PLP-Ni species (derived from the fluorescence values at 340 nm) plotted against the total added Ni(II) concentration. The fit was achieved with n=2.

Figure 62 :

 62 Figure 62: Microcalorimetric titration of PLP by copper sulfate, heat flow against Molar Ratio. Successive injections at 20°C, of 2 µl of a 600 µM solution of Copper sulfate in 50mM MOPS, 300mM NaCl were made on 50 µM PLP in 50mM MOPS, 300mM NaCl .

Figure 63 :Figure 64 :

 6364 Figure 63: Microcalorimetric titration of PLP by nickel sulfate, heat flow against Molar ratio. Successive injections at 20°C, of 2 µl of a 600 µM solution of nickel sulfate in 50mM MOPS, 300mM NaCl were made on 50 µM PLP in 50mM MOPS, 300mM NaCl.

Figure 65 :

 65 Figure 65: Microcalorimetric titration of iron free PLP by zinc sulfate, heat flow versus Molar Ratio. Successive injections at 20°C, of 2 µl of a 300 µM solution of zinc sulfate in 50mM MOPS, 300mM NaCl were made on 24 µM PLP, 50mM MOPS, 300mM NaCl.

Figure 66 :

 66 Figure 66: Microcalorimetric titration of iron free PLP by copper sulfate, heat flow against time. Successive injections at 20°C, of 2 µl of a 300 µM solution of copper sulfate in 50 mM MOPS and 300 mM NaCl were made on 24 µM PLP in 50 mM MOPS and 300 mM NaCl.

Figure 67 :

 67 Figure 67: Microcalorimetric titration of iron free PLP by Nickel nitrate, heat flow against molar ratio. Successive injections at 20°C, of 2 µl of a 300 µM solution of nickel nitrate were made on 24 µM PLP in 50 mM MOPS and 300 mM NaCl.

Figure 68 :

 68 Figure 68: TYCHO measurements of PLP-metal complexes upon addition of 10 3 equiv. of EDTA. Orange: PLP-CuNO3; Purple: PLP; Pink: PLP-NiNO3; Blue: PLP-ZnSO4; Green: PLP-CuSO4.

Figure 69 :

 69 Figure 69: EPR spectrum of PLP-Cu protein measured at 10 K at a 9.3 GHz frequency; 80 µM PLP-Cu in 50mM MOPS buffer, 300 mM NaCl, 15% Glycerol.

Figure 70 :

 70 Figure 70: TYCHO measurements of PLP before and after the copper addition. Purple: 10 µM PLP; orange: 30 µM iron free PLP (30 mM EDTA); Pink: 30 µM iron free PLP (30 mM EDTA) +2 equiv of Copper sulfate.

Figure 71 :

 71 Figure 71: TYCHO measurements of PLP before and after the addition of 10 equiv. of copper. Purple: 10 µM PLP; orange: 30 µM iron free PLP with 30 mM EDTA; Pink: 30 µM iron free PLP with 30 mM EDTA +10 equiv. Copper.

Scheme 18 :

 18 Scheme 18: Strategy for the design of the chimeric PMI-PLP enzyme. Searching for the suitable linking point.

Figure 72 :

 72 Figure 72: Visualizing and analyzing normal modes of vibration of the PMI protein

Figure 73 :

 73 Figure 73: A) Overlay of the crystal structure of PMI (in gold) and modeled structure of the chimeric enzyme (in blue); B) Overlay of the structure of PLP (in gold-published model 7 ) and modeled structure of the chimeric enzyme (in blue).

Figure 74 :

 74 Figure 74: Modeled structure of the chimeric PMI-PLP enzyme as predicted by I-TASSER (left), and by MODELLER server (right). Fragment of PMI in red; fragment of PLP in yellow.

Figure 75 :

 75 Figure 75: Predicted structures of the chimera 2. Left: prediction with MODELLER. Right: prediction with I-TASSER server. In red: PMI; in yellow: PLP

Scheme 19 :

 19 Scheme 19: Construction of the expression vector of the chimeric enzyme. In blue: the sequence encoding for the PMI's active site. In red: the sequence encoding for the PLP's active site.

Figure 78 :

 78 Figure 78: Coomassie blue stained SDS page electrophoresis of pellet fractions after the expression of the His6 tagged Chimera 1 by addition of different concentrations of IPTG.

Figure 79 :

 79 Figure 79: Western blot image of the His6 -Chimera 1.

Figure 80 :

 80 Figure 80: A) Coomassie blue stained SDS-PAGE electrophoresis of collected fractions of the first purification on an Ni-NTA column of the chimeric enzyme. B) Western Blot image. In Both cases, lanes: 1, 2 and 3: Flow through; 4, 5 and 6: Washed fractions; 7, 8 and 9: Eluted fractions; L: Protein Ladder

Figure 81 :

 81 Figure 81: Amino acid (aa) sequence alignment of Chimera 1 with the synthetic Chimera 2.

Figure 82 :

 82 Figure 82: Analysis by agarose gel electrophoresis followed by ETBr staining of the result of the digestion of the pET-28b plasmid by BamHI and PstI. From left to right: First lane: Molecular adder; Second lane: Intact plasmid; Third lane: band at about 6 kB corresponding to the digested plasmid.

  left) was observed in all pellet lanes at a MW of about 60 kDa corresponding to different IPTG concentrations. However, no band was observed in the first lane where the protein was not induced. This first assay shows that 0,1 mM IPTG is enough to induce the production of the Chimera 2 in BL21 E.coli.Upon 18 hours of induction, a band with high intensity than the one before was observed in all pellet lanes (lanes 2 and 3, Figure83right) at a MW of about 61 kDa, which reflected the expression of Chimera 2. In contrast, no protein band could be detected in the supernatant fractions (lane 1, Figure83 right). This means that the chimera 2 is better expressed at low temperature for overnight incubation.

Figure 83 : 1 :

 831 Figure 83: SDS-PAGE electrophoresis followed by Coomassie blue staining of supernatant fractions and pellets after the expression of the Chimera 2 by addition of different concentrations of IPTG. Left: After 5 hours of induction; Right: Overnight expression; Lane 1: Supernatant fraction; Lanes 2 and 3: Pellet fractions L: Protein Ladder.

Figure 84 :

 84 Figure 84: Characterization of the expression of the chimeric 2 before cell lysis. Cultures were made in LB and TB media supplemented with Kanamycin. L: Ladder; Lanes 1 to 4: LB medium and Lanes 5 to 8: TB medium. Lanes: 1&5: No induction -Lanes 2&6: 0.1 mM IPTG -Lanes 3 & 7: 0.2 mM IPTG -Lanes 4&8: 0.4 mM IPTG

Figure 85 :

 85 Figure 85: SDS-PAGE electrophoresis followed by Coomassie blue staining of supernatant fractions and pellets after the cell lysis of the expressed Chimera 2. Lanes 1 to 4: in LB medium Lanes 5 to 8: in TB medium. Lanes 1, 3, 5 and 7: Soluble supernatant fractions; Lanes 2, 4, 6 and 8: insoluble fractions. L: Protein Ladder.

Figure 86 :Figure 87 :

 8687 Figure 86: SDS-PAGE electrophoresis followed by Coomassie blue staining of supernatant fractions and pellets after the cell lysis of the expressed proteins. S: Supernatant -P: Pellet -Ch1: Chimera 1 -Ch2: Chimera 2

  chaperone teams are known to have different and cooperative functions:102,104,105 On the first hand, the lack of one chaperone team can increase the requirement for the other and vice versa. On the other hand, a protein can be stabilized in substantially aggregated forms by DnaK-DnaJ mutations while it can be destabilized by GroELand GroES mutations.102 In addition, these proteins can enhance the stability, the folding and the disaggregation of different individual proteins.102,104,105

Figure 88 :

 88 Figure 88: Structure of chaperone coexpression plasmid pG-KJE6. Ori: replication origin of pACYC184; cat: chloramphenicol acetyltransferase gene; Pzt-1p: promoter; tetR: tetR repressor gene; araBp/o: araB promoter-operator; araC: araC repressor gene. 102

Figure 89 :

 89 Figure 89: Analysis by SDS PAGE to characterize the expression of the chimera 2 coexpressed with chaperone proteins in LB and TB medium. Lanes: 1, 2, 3 and 4: LB medium -5, 6, 7 and 8: TB medium. 1 and 5: = supernatant, no induction -2 and 6: inclusion body, no induction -3 and 7: supernatatnt, induction -4 and 8: inclusion body, induction -L: Ladder

Figure 90 :

 90 Figure 90: Western blot image using anti his tag antibody blot to characterize the expression of the chimera 2 co-expressed with chaperone proteins in LB and TB medium. Lanes 1, 2, 3 and 4: supernatant fractions. Lanes 5, 6, 7 and 8: inclusion bodies fractions.

Scheme 22 :

 22 Scheme 22: Construction of the expression vector of Half PMI. P1: Forward primer; P2: Reverse primer.

Figure 91 :

 91 Figure 91: Coomassie blue stained SDS-PAGE electrophoresis of the collected fractions after the purification on an Ni-NTA agarose column of His6 tagged Half PMI. L: Ladder; Lane 1: Washed fraction, Lanes 2 and 3: Elution fractions

Figure 93 :

 93 Figure 93: Western Blot image using anti His6 antibody of the fraction eluted during the purification of the Half His6 PMI protein.

Scheme 23 :

 23 Scheme 23: Construction of the expression vector of the His6 -Half PLP by the Gibson assembly method.

Figure 94 :

 94 Figure 94: Western Blot image using anti His6 antibody of the collected elution fractions of the first purification on an Ni-NTA column of the Half PLP protein.

  enzyme expression vector yielded PLP comprising a His6 tag co-expressed with a second protein formed of a portion of LacZ fused to His6-PLP. The coexpression was confirmed by an anti His6 western blot which revealed two bands instead of a single band. To avoid this co-expression, the expression plasmid was edited by deleting the remaining portion of LacZ. The expression plasmid was digested and a site for specific hydrolysis by TEV protease was successfully introduced flanked by the sequences of the enzyme and that of the His6-tag. Purified and sequenced plasmid DNA containing the insert sequence was used to transform BL21(DE3) for expressing the protein. After expression and purification on an Ni-NTA column, the enzyme was separated from the His6 tag by digestion with TEV protease. Following the digestion, the TEV protease with its His6 tag was easily removed from the cleavage reaction by purification on a Ni-NTA affinity column. The produced PLP contains one Fe(II) cation per enzyme molecule, therefore the removal of Fe(II) from the active site was performed by incubating the protein with 10 3 equiv. EDTA. To prepare the artificial metalloprotein, Ni 2+ , Zn 2+ and Cu 2+ were added to the iron free protein. A relatively good dissociation constant (KD = 0.5 µM) was measured by fluorescence quenching upon addition of increasing amounts of copper sulfate. The EPR spectrum of the PLP-Cu complex confirmed the presence of a single copper species. TYCHO results showed that the thermal Chapter 6: General Conclusion and Future work denaturation occured at 75°C and that high concentrations of EDTA and of copper sulfate could alter the folding of PLP. Hence, PLP was successfully transformed into an artificial copper(II)-containing protein. Quercetin is relatively bigger than the azachalcone and cyclopentadiene and since copper-containing PLP catalyzed quercetin oxidation we expect that it will also catalyze the Diels-Alder cycloaddition after future works.Since the two starting enzymes were expressed, purified, transformed into artificial metalloproteins and characterized by different techniques, we proceeded to develop an artificial chimeric metalloenzyme that possesses multiple active sites by the fusion of catalytic domains of these two related enzymes. First, the strategy we followed to prepare the PMI-PLP bicupin chimera was based on structural modeling studies and on the determination of the linking point between the two catalytic domains of PMI and PLP. elNémo was used to simulate the low frequency normal modes of vibration of PMI and of PLP. The movements of all atoms/domains in PMI and PLP were thus studied and visualized. The 1 st and the 2 nd mode of vibration of PMI show that the two cupin domains vibrate independently. The 3 rd normal mode of vibration originates at the center of the linker of the cupin domains thus the association of the two different cupin domains can occur by fusion at the center of their interdomain linkers. The sequences encoding for the PMI active site and the PLP active site were cloned by PCR.

Scheme 24 :

 24 Scheme 24: Reversible isomerization of D-mannose-6-phosphate (M6P) to D-fructose-6phosphate (F6P) catalyzed by the chimera.

Scheme 26 :

 26 Scheme 26: Two reactions in cascade catalyzed by heterobinuclear Cu(II) -Zn(II) containing chimera.

  chloride) and eluted by adding ELU-EF. The plasmid DNA was precipitated by adding isopropanol. After centrifugation at 15000 g for 40 min at 4 °C, the supernatant was discarded and 70 % ethanol was added to the pellet. After centrifugation at 15000 g for 15 min at 4 °C, the supernatant was discarded and the pellets were dissolved in an appropriate volume of sterile nuclease-free water.DNA concentration (3.2 μg/μL) and purity (A260 = 64,963; A280 = 34,621; A260/A280 = 1,88) were determined by measuring the UV-vis absorption spectra of the samples on a nanodrop spectrophotometer (Thermo Scientific) apparatus.

DNA concentration ( 70

 70 Scheme 27: Sequence of Nhe1 recognition site and its restriction products once DNA has been digested.

  gentle tapping and was incubated in an ice bath for 30 minutes. The cells were transferred in a 45 °C water bath for exactly 45 seconds and then back in ice for 2 minutes. 960 µl of SOC medium (Sigma) were added and the cells were placed on a shaker incubator (225 rpm) at 37 °C for 1 hour. 100 μl of the mixture containing the transformed cells were coated onto LB agar plates with ampicillin (100 μg/mL) and the plates were incubated at 37 °C overnight (225 rpm).

A

  polypropylene round bottom tubes, mixed by gentle tapping and incubated in ice for 30 minutes. The cells were transferred in a 42 °C water bath for exactly 45 seconds and then back to ice for 2 minutes. 650 µl of SOC medium (NEB) were added and the resulting mixture was placed on a shaker incubator (225 rpm) at 37 °C for 1 hour. 100 μl of the mixture containing the transformed cells were coated onto LB agar plates with kanamycin (50 μg/mL) and incubated at 37 °C overnight (225 rpm). PCR mixture was prepared using the Thermofisher Taq polymerase starter kit, by adding 2.5 µl Taq polymerase reaction Buffer, 4 µl MgCl2 (Ci= 25 mM), 2.5 µl dNTPs (Ci=2mM), 25 µM primers, 1 µl nuclease free Taq DNA Polymerase (1U/µl), 15 µl water and finally a small amount of each colony on the tip of a wooden toothpick was added.The reaction was gently mixed and the PCR tubes (50 µl) were transferred from ice to the PCR machine. The PCR was carried out in a Perkin-Elmer Thermocycler starting with Materials and methods two minutes at 95 °C for Taq activation followed by 40 2-minutes cycles involving 30 seconds at 95 °C for DNA denaturation, 30 seconds at 48 °C for hybridization and one minute at 72 °C for extension.

  5' ATG GCC CAA CGG GAA ATT CTA TCC ATC ACG ACC GGC 3' and P2: 5' AGC TFT TTC CTG TGT GAA ATT GTT ATC CGC TCA CAA TTC CAC ACA ACA TAC GAG 3') in an applied biosystem thermocycler (StepOne Plus). It started with 30 seconds at 98 °C for Q5 Hot start high fidelity polymerase activation, followed by 25 3-minutes cycles involving 10 seconds at 98 °C for DNA denaturation, 20 seconds at 65 °C for hybridization, 30 seconds at 72 °C for extension and two minutes at 72 °C for final extension. The presence of the PCR amplicon and the size of the product were determined by electrophoresis alongside a DNA size marker on a precast 1.2% agarose gel.

  two primers: for PMI amplification: P1: 5' CGA CTT CGC ATA AGG ATC CGA CCT CGA GAT CTG 3' and P2: 5' TAG GCC GGC CCA TTT TCT GTT TTT CAA CGC TTT CGT AGC T 3', for PLP amplification: (P1: 5' ACA GAA AAT GGG CCG GCC TAC ATC CG 3' and P2: 5' CG ATC CTT ATG CGA AGT CGC GGT AAC C 3'). Each PCR started with 30 seconds at 98°C for Taq polymerase activation. Then for PMI, by 25 4-minutes and 50-seconds cycles were performed including 10 seconds at 98°C for DNA denaturation, 20 seconds at 66°C for hybridation, 140 s at 72°C for PMI for extension and two minutes at 72 °C for primer final extension. For PLP, 25 2-minutes and 45-seconds cycles were performed including 10 seconds at 98°C for DNA denaturation, 20 seconds at 64°C for hybridation, 15 s at 72°C for PLP for extension and two minutes at 72 °C for primer final extension.

  Proteins present in the samples could be separated by SDS-PAGE and visualized by Coomassie brilliant blue staining (InstantBlue from Expedeom). 60 μL commercial 2x Laemmli (4 % SDS, 20 % glycerol, 10 % 2-mercaptoethanol, 0.004 % bromophenol blue and 0.125 M Tris HCl, pH 6.8, from Sigma) were mixed with 60 μL sample and the resulting solution was boiled for 5 min. 15 μL aliquots of the final denatured samples were deposited in wells of a 12 % polyacrylamide gel. The addition of a mixture of control proteins of known molecular weight (SeeBleu -Thermofisher / Broad Range) in a control well, to make a molecular weight ladder, allowed the verification of the expected protein molecular weights. Migration was performed by vertical electrophoresis in Turbo reducing running buffer (0.6 M MOPS, 2% SDS, 1.2 M Tris base from C.B.S.) at 180 V (requiring about 110 mA) until the visible bromophenol blue reached the top of the gel. The gel was stained by incubation in Coomassie brilliant blue solution for 30 min, washed 5 times with water at 15 min intervals, and fixed with 5 % acetic acid. The amount and the purity of the proteins was estimated from the intensity of the bands revealed on the gel. -Western Blot: After SDS-PAGE the protein bands were electro transferred horizontally onto a nitrocellulose membrane 70 minutes in migration buffer (25mM Trizma base, 192 mM Glycine, 20% ethanol, pH= 8.4) at 72V (requiring 220 mA). To avoid false positives due to non-specific staining of the nitrocellulose membrane by antibodies, the membrane was soaked in a 3 % BSA solution supplemented with the primary anti-His6 mouse Materials and methods antibody (dilution:1:250) and incubated at 4 °C overnight with shaking. The next day, the membrane was washed three times with PBS every 15 minutes and was then incubated with the secondary anti-mouse antibody conjugated with horseradish peroxidase HRP (0.3 µg/mL) in the presence of 20 mL PBS 1X at 4 °C for 2 hours at 4 °C with shaking. The membrane was finally washed 3 times with PBS every 15 minutes.

Figure 1 :

 1 Figure 1: A) 3D structure of PMI B) Swiss model of PLP .......................................................... Figure 2: Predicted structure of PMI-PLP chimeric enzyme. In red: catalytic domain of PMI; in yellow: catalytic domain of PLP. ........................................................................................Figure 3: Superposition of PMI and PLP.......................................................................................Figure 4: Three strategies for site-specific introduction of transition metals or their complexes into in protein hosts (L = ligand; M = transition metal) ...................................Figure 5: Production of enzymes with an optimized catalytic activity following the directed evolution strategy. 23 ............................................................................................................Figure 6: Crystal structure of ACCO proving that it belongs to the cupin superfamily of enzymes and revealing its iron(II) containing active site. ..................................................Figure7: Two fundamental types of chimeric proteins: Type 1: The two proteins or protein subunits are united end-to-end and usually linked by a linker; type 2: the amino acids from both donors are interspersed in the fusion protein product. 8 .......... Figure8: Common strategies for the construction of fusion proteins.[START_REF] Yu | Synthetic fusion protein design and applications[END_REF] ........................... Figure9: The engineering of a chimeric enzyme comprising two cellulases, CelA and SSO1949 using the 'domain insertion' approach. The SCHEMA tool was used to find the insertion sites on CelA.[START_REF] Yu | Synthetic fusion protein design and applications[END_REF] .............................................................................................................Figure 10: Structures showing the conformational changes of CaM in the closed state (pdb: 1CFD) and bound to Ca 2+ (pdb: 1CLL). 45 ............................................................................Figure 11: Schematic representation of the recombinant TNF -IL-12 protein comprising the IL-12, TNF and scFv human antibody. 48 .........................................................Figure 12: Characteristic structure of cupin proteins showing two conserved 2-strands domains separated by a less conserved inter-motif region containing another 2--strands and a loop. ....................................................................................................................... Figure 13: Crystal structure of ACCO showing its unique cupin domain and revealing its Fe 2+ containing active site.............................................................................................................Figure 14: Crystal structure of Human Pirin (PDB: 1J1L) revealing its Fe 2+ containing site. ..............................................................................................................................................................Figure 15: An example of multicupin: Human homogentisate dioxygenase (HGO). ....Figure 16: Schematic representation of the structure of PMI showing a large interdomain between two conserved barrels containing Zinc in the active site located on the C-terminal side................................................................................................................................Figure 17: 3D structure of PMI showing a central β-barrel catalytic domain flanked by helical domain on one side and a N-terminal β-barrel domain on the other side. ......52 Figure 18: View of the metal binding site in PMI (pdb : 1PMI). Interatomic distances are given in Å. .........................................................................................................................................Figure 19: Crystal structure of His6-PMI coordinated to the inhibitor 5PAHz. ...............

Figure 54 :

 54 Figure 49: Western Blot image of His6-PLP. Lanes: 1-Protein Ladder; 2-Flow through; 3-Washed fraction; 5-6-7-8-Eluted fractions. ...........................................................................Figure 50: Right, Coomasie blue stained SDS-PAGE electrophoresis of PLP upon cleavage of the His6 tag (on the right) followed by Western Blot image using Anti His primary antibody (on the left). L: Ladder; Lane 1: His6-PLP + TEV; Lane 2: Tag free PLP ......................................................................................................................................................................Figure 51: Fluorescence titration of the binding of Cu(II) to PLP with 10 equiv. EDTA upon excitation at 280 nm. No significant quenching of fluorescence was observed suggesting the remaining of iron cation in the active site of PLP. ......................................Figure 52: TYCHO measurements of 10 µM native PLP-Fe in black and 10 µM PLP iron free in pink. .............................................................................................................................................. Figure 53: TYCHO measurements of 10 µM native PLP in orange and 10 µM PLP iron free upon addition of 10 mM EDTA in purple. ...........................................................................Figure 54: Titration of the fluorescence of native PLP upon the addition of increasing amounts (0.2-55 equiv.) of Cu(II) (left) and Zn(II) (right) (excitation at 280 nm). A nonsignificant quenching of the signal was seen in both cases. Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP tag free. .........Figure 55: Left: Titration of the fluorescence of native PLP upon the addition of increasing amounts of Ni 2+ (excitation at 280 nm). Right: Fluorescence of PLP at 320 nm as function of increasing equivalents of added Ni 2+ . Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP. ..........................Figure 56: Quenching of the fluorescence of iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Cu(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP. ............... Figure 57: Curve showing the increase of Cu-PLP concentration, derived from the fluorescence values at 340 nm, as a function of the total concentration of added Cu(II). .......................................................................................................................................................... Figure 58: Quenching of the fluorescence of native iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Zn(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 1.25 µM PLP. ............Figure 59: Quenching of the fluorescence of native iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Ni(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 1.25 µM PLP. ............Figure 60: Fluorescence of PLP at 320 nm as function of increasing equivalents of metal cations added. PLP-Cu(II) complex in blue, PLP-Ni(II) complex in orange and PLP-Zn(II) complex in gray. ................................................................................................................Figure 61: A) concentration of PLP-Ni (derived from the fluorescence values at 340 nm) plotted against the total added Ni(II) concentration. The fit was achieved with n=1. B) total concentration of different PLP-Ni species (derived from the fluorescence

Figure 55 :Figure 56 :Figure 57 :Figure 59 :

 55565759 Figure 49: Western Blot image of His6-PLP. Lanes: 1-Protein Ladder; 2-Flow through; 3-Washed fraction; 5-6-7-8-Eluted fractions. ...........................................................................Figure 50: Right, Coomasie blue stained SDS-PAGE electrophoresis of PLP upon cleavage of the His6 tag (on the right) followed by Western Blot image using Anti His primary antibody (on the left). L: Ladder; Lane 1: His6-PLP + TEV; Lane 2: Tag free PLP ......................................................................................................................................................................Figure 51: Fluorescence titration of the binding of Cu(II) to PLP with 10 equiv. EDTA upon excitation at 280 nm. No significant quenching of fluorescence was observed suggesting the remaining of iron cation in the active site of PLP. ......................................Figure 52: TYCHO measurements of 10 µM native PLP-Fe in black and 10 µM PLP iron free in pink. .............................................................................................................................................. Figure 53: TYCHO measurements of 10 µM native PLP in orange and 10 µM PLP iron free upon addition of 10 mM EDTA in purple. ...........................................................................Figure 54: Titration of the fluorescence of native PLP upon the addition of increasing amounts (0.2-55 equiv.) of Cu(II) (left) and Zn(II) (right) (excitation at 280 nm). A nonsignificant quenching of the signal was seen in both cases. Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP tag free. .........Figure 55: Left: Titration of the fluorescence of native PLP upon the addition of increasing amounts of Ni 2+ (excitation at 280 nm). Right: Fluorescence of PLP at 320 nm as function of increasing equivalents of added Ni 2+ . Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP. ..........................Figure 56: Quenching of the fluorescence of iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Cu(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP. ............... Figure 57: Curve showing the increase of Cu-PLP concentration, derived from the fluorescence values at 340 nm, as a function of the total concentration of added Cu(II). .......................................................................................................................................................... Figure 58: Quenching of the fluorescence of native iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Zn(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 1.25 µM PLP. ............Figure 59: Quenching of the fluorescence of native iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Ni(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 1.25 µM PLP. ............Figure 60: Fluorescence of PLP at 320 nm as function of increasing equivalents of metal cations added. PLP-Cu(II) complex in blue, PLP-Ni(II) complex in orange and PLP-Zn(II) complex in gray. ................................................................................................................Figure 61: A) concentration of PLP-Ni (derived from the fluorescence values at 340 nm) plotted against the total added Ni(II) concentration. The fit was achieved with n=1. B) total concentration of different PLP-Ni species (derived from the fluorescence

Figure 60 :

 60 Figure 49: Western Blot image of His6-PLP. Lanes: 1-Protein Ladder; 2-Flow through; 3-Washed fraction; 5-6-7-8-Eluted fractions. ...........................................................................Figure 50: Right, Coomasie blue stained SDS-PAGE electrophoresis of PLP upon cleavage of the His6 tag (on the right) followed by Western Blot image using Anti His primary antibody (on the left). L: Ladder; Lane 1: His6-PLP + TEV; Lane 2: Tag free PLP ......................................................................................................................................................................Figure 51: Fluorescence titration of the binding of Cu(II) to PLP with 10 equiv. EDTA upon excitation at 280 nm. No significant quenching of fluorescence was observed suggesting the remaining of iron cation in the active site of PLP. ......................................Figure 52: TYCHO measurements of 10 µM native PLP-Fe in black and 10 µM PLP iron free in pink. .............................................................................................................................................. Figure 53: TYCHO measurements of 10 µM native PLP in orange and 10 µM PLP iron free upon addition of 10 mM EDTA in purple. ...........................................................................Figure 54: Titration of the fluorescence of native PLP upon the addition of increasing amounts (0.2-55 equiv.) of Cu(II) (left) and Zn(II) (right) (excitation at 280 nm). A nonsignificant quenching of the signal was seen in both cases. Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP tag free. .........Figure 55: Left: Titration of the fluorescence of native PLP upon the addition of increasing amounts of Ni 2+ (excitation at 280 nm). Right: Fluorescence of PLP at 320 nm as function of increasing equivalents of added Ni 2+ . Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP. ..........................Figure 56: Quenching of the fluorescence of iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Cu(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP. ............... Figure 57: Curve showing the increase of Cu-PLP concentration, derived from the fluorescence values at 340 nm, as a function of the total concentration of added Cu(II). .......................................................................................................................................................... Figure 58: Quenching of the fluorescence of native iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Zn(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 1.25 µM PLP. ............Figure 59: Quenching of the fluorescence of native iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Ni(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 1.25 µM PLP. ............Figure 60: Fluorescence of PLP at 320 nm as function of increasing equivalents of metal cations added. PLP-Cu(II) complex in blue, PLP-Ni(II) complex in orange and PLP-Zn(II) complex in gray. ................................................................................................................Figure 61: A) concentration of PLP-Ni (derived from the fluorescence values at 340 nm) plotted against the total added Ni(II) concentration. The fit was achieved with n=1. B) total concentration of different PLP-Ni species (derived from the fluorescence

Figure 61 :

 61 Figure 49: Western Blot image of His6-PLP. Lanes: 1-Protein Ladder; 2-Flow through; 3-Washed fraction; 5-6-7-8-Eluted fractions. ...........................................................................Figure 50: Right, Coomasie blue stained SDS-PAGE electrophoresis of PLP upon cleavage of the His6 tag (on the right) followed by Western Blot image using Anti His primary antibody (on the left). L: Ladder; Lane 1: His6-PLP + TEV; Lane 2: Tag free PLP ......................................................................................................................................................................Figure 51: Fluorescence titration of the binding of Cu(II) to PLP with 10 equiv. EDTA upon excitation at 280 nm. No significant quenching of fluorescence was observed suggesting the remaining of iron cation in the active site of PLP. ......................................Figure 52: TYCHO measurements of 10 µM native PLP-Fe in black and 10 µM PLP iron free in pink. .............................................................................................................................................. Figure 53: TYCHO measurements of 10 µM native PLP in orange and 10 µM PLP iron free upon addition of 10 mM EDTA in purple. ...........................................................................Figure 54: Titration of the fluorescence of native PLP upon the addition of increasing amounts (0.2-55 equiv.) of Cu(II) (left) and Zn(II) (right) (excitation at 280 nm). A nonsignificant quenching of the signal was seen in both cases. Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP tag free. .........Figure 55: Left: Titration of the fluorescence of native PLP upon the addition of increasing amounts of Ni 2+ (excitation at 280 nm). Right: Fluorescence of PLP at 320 nm as function of increasing equivalents of added Ni 2+ . Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP. ..........................Figure 56: Quenching of the fluorescence of iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Cu(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 2.5 µM PLP. ............... Figure 57: Curve showing the increase of Cu-PLP concentration, derived from the fluorescence values at 340 nm, as a function of the total concentration of added Cu(II). .......................................................................................................................................................... Figure 58: Quenching of the fluorescence of native iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Zn(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 1.25 µM PLP. ............Figure 59: Quenching of the fluorescence of native iron free PLP upon the addition of increasing amounts (0.2-55 equiv.) of Ni(II) (excitation at 280 nm). Measurements were performed in 50 mM MOPS, 300mM NaCl, pH = 7,6 using 1.25 µM PLP. ............Figure 60: Fluorescence of PLP at 320 nm as function of increasing equivalents of metal cations added. PLP-Cu(II) complex in blue, PLP-Ni(II) complex in orange and PLP-Zn(II) complex in gray. ................................................................................................................Figure 61: A) concentration of PLP-Ni (derived from the fluorescence values at 340 nm) plotted against the total added Ni(II) concentration. The fit was achieved with n=1. B) total concentration of different PLP-Ni species (derived from the fluorescence

Figure 87 :Figure 88 :Figure 89 :Figure 90 :Figure 97 :

 8788899097 Figure 87: Western Blot image. L: Ladder -S: Supernatant -P: Pellet -Ch1: Chimera 1 -Ch2: Chimera 2 ..................................................................................................................................117 Figure 88: Structure of chaperone coexpression plasmid pG-KJE6. Ori: replication origin of pACYC184; cat: chloramphenicol acetyltransferase gene; Pzt-1p: promoter; tetR: tetR repressor gene; araBp/o: araB promoter-operator; araC: araC repressor gene. 102 ....................................................................................................................................................118 Figure 89: Analysis by SDS PAGE to characterize the expression of the chimera 2 coexpressed with chaperone proteins in LB and TB medium. Lanes: 1, 2, 3 and 4: LB medium -5, 6, 7 and 8: TB medium. 1 and 5: = supernatant, no induction -2 and 6: inclusion body, no induction -3 and 7: supernatatnt, induction -4 and 8: inclusion body, induction -L: Ladder .............................................................................................................119 Figure 90: Western blot image using anti his tag antibody blot to characterize the expression of the chimera 2 co-expressed with chaperone proteins in LB and TB medium. Lanes 1, 2, 3 and 4: supernatant fractions. Lanes 5, 6, 7 and 8: inclusion bodies fractions. ...................................................................................................................................119 Figure 91: Coomassie blue stained SDS-PAGE electrophoresis of the collected fractions after the purification on an Ni-NTA agarose column of His6 tagged Half PMI. L: Ladder; Lane 1: Washed fraction, Lanes 2 and 3: Elution fractions ...............................124 Figure 92: SDS-PAGE electrophoresis followed by Coomassie blue staining of the elution fractions collected after the purification on an Ni-NTA agarose column of His6-Half PMI using the BL21 E.coli pLysS strain. .....................................................................124 Figure 93: Western Blot image using anti His6 antibody of the fraction eluted during the purification of the Half His6 PMI protein. ............................................................................125 Figure 94: Western Blot image using anti His6 antibody of the collected elution fractions of the first purification on an Ni-NTA column of the Half PLP protein. ........127 Figure 95: Vector map of pRSET-C showing its important features and containing PMI and His6 tag flanking TEVc/site. .........................................................................................................168 Figure 96: Vector map of pUC18 showing its important features and containing PLP and His6 tag flanking TEVc/site. .........................................................................................................169 Figure 97: Vector map of pRSET-C showing its important features and containing the first chimeric PMI-PLP protein and His6 tag flanking TEVc/site. ............................................170

  Table of contents -TYCHO : ..........................................................................................................................150 -Peroxidase activity: .....................................................................................................150 Bibliography ..........................................................................................................................................152 List of Figures ...

Table 1 :

 1 Features Different strategies to construct artificial metalloenzymes: Initial challenges to construct artificial metalloenzymes have been explored by Kaiser and co-workers 3 as well as Wilson and Whitesides 18 since the late 1970's. Different strategies have thus been developed[START_REF] Di Meo | αRep A3: A Versatile Artificial Scaffold for Metalloenzyme Design[END_REF][START_REF] Mahy | Current Applications of Artificial Metalloenzymes and Future Developments[END_REF] such as:

and limitations of natural metalloenzymes and chemically synthesized models, in comparison to those of artificial metalloenzymes.

[START_REF] Steinreiber | Artificial metalloenzymes as selective catalysts in aqueous media[END_REF]

Chapter 1: State of the art 1.1.1.

  [START_REF] Yu | Synthetic fusion protein design and applications[END_REF]

resulting in a one-pot carbon-carbon bond synthesis technique (scheme 7) with 20-fold increase in the aldol reaction rate. 28,41

  In 2012, Choi et al. engineered a chimeric enzyme composed of Cytochrome P450

	Chapter 1: State of the art
	 In 2010, a chimeric protein was constructed by Wang et al. combining
	arabinosidase, xylanase and xylosidase. This trifunctional protein promoted the
	degradation of complex agricultural byproducts with an obvious increase in
	non-natural amino acids with unusual functional groups, such as azide, ketone, and activity. 28,42
	phosphate, into target proteins via stop codon suppressor tRNA and in vitro translation
	technology would provide a new level of flexibility in the creation of post-translational  In 2012, Choudhoury et al. developed an artificial chimeric enzyme via the fusion of
	protein fusions. 40 an RNA cleavage domain and Pumilio/fem-3 mRNA-binding factors. The two
	domains were linked via the tandem strategy, and the RNA endonucleases that
	1.2.4. Applications of the fusion proteins: resulted were able to detect RNA substrates and cleave them efficiently near their
	binding sites. 28,43
	Artificial fusion proteins have been used in different technologies, so they have a broad
	range of applications. 28 Some applications are described below: 
	1.2.4.1. Multifunctional biocatalytic activities:
	Engineered fusion enzyme may lead to improved capabilities vs the parent proteins as
	merging functional properties from different enzyme moieties in a single one,
	facilitates the channeling of the substrates in a sequential process, reduces the effective
	reaction volume, and improves electron transfer. 28
	 In 2010, Iturrate et al. engineered a bifunctional enzyme composed of
	dihydroxyacetone (DHA) kinase and fructose-1,6-bisphosphate (FBP) aldolase. DHA
	was transformed to the unstable dihydroxyacetone phosphate (DHAP) by the DHA
	kinase part of the chimera. DHAP was then used as a substrate in the aldol
	condensation reaction catalyzed by (FBP) aldolase part of the chimera to form
	phosphorylated aldol adduct, Scheme 7: Reaction catalyzed by the bifunctional enzyme composed of DHA kinase and FBP
	aldolase. 41 Benzyloxyacetaldehyde, acetaldehyde and 3-(methylthio)propionaldehyde were
	the assayed aldehydes.

blob

  Physarum polycephalum to stress was discovered byBernier et al. In 1987.

	Chapter 1: State of the art
	activity. Part of this sequence similarity comes from a common decapeptide sequence
	PH(I/T)HPRAIEI named the Germin Box. In 1998, Dunwell and Gane proved that this
	box was part of a much larger conserved region in numerous proteins, that they named
	the cupin domain.
	Spherulins have about 44% sequence similarity with germin 53 but show no ox-ox

Table 2 :

 2 Classification of Cupins according to the Protein database.

Table 3 :

 3 Different organisms providing different types of PMI.

	PMI Type I	PMI Type II	PMI Type III
	Saccharomyces cerevisiae	Escherichia coli	Rhizobium meliloti
	Aspergillus nidulans	Salmonella typhimurium
	Candida albicans	Acetobacter xylinum
	Leishmania mexicana	Acinetobacter calcoaceticus
	Escherichia coli	Helicobacter pylori
	Salmonella enterica	Pseudomonas aeruginosa
	Cryptococcus neoformans	Rhodospirillum rubrum
	Caenorhabditis elegans	Xanthomnas campestris
	Streptococcus mutans
	Homo sapiens

2.2.1.1. Type I PMIs:

Table 4 :

 4 Various chemicals and enzymes used in the test set up to measure the specific PMI activity. PGI and G6PDH volumes were added so that their final concentration was 100 fold

		higher than that of PMI.
		0.1 M M6P in water	20 µL
		0.5 M MgCl2 in water	10 µL
		40 mM NADP + in water	10 µL
		V(PGI/100)	40 µL
		V(G6PDH/100)	100 µL
		12 µM PMI	30, 60, 90 or 120 µL
		50 mM MOPS, pH= 7.1	up to 1mL
	-	Zinc removal:

  ...................................................... Figure 2: Predicted structure of PMI-PLP chimeric enzyme. In red: catalytic domain of PMI; in yellow: catalytic domain of PLP. ........................................................................................Figure 3: Superposition of PMI and PLP.......................................................................................Figure 4: Three strategies for site-specific introduction of transition metals or their complexes into in protein hosts (L = ligand; M = transition metal) ...................................Figure 5: Production of enzymes with an optimized catalytic activity following the directed evolution strategy. 23 ............................................................................................................ Figure 6: Crystal structure of ACCO proving that it belongs to the cupin superfamily of enzymes and revealing its iron(II) containing active site. ..................................................Figure7: Two fundamental types of chimeric proteins: Type 1: The two proteins or protein subunits are united end-to-end and usually linked by a linker; type 2: the amino acids from both donors are interspersed in the fusion protein product. 8 .......... Figure8: Common strategies for the construction of fusion proteins.[START_REF] Yu | Synthetic fusion protein design and applications[END_REF] ........................... Figure9: The engineering of a chimeric enzyme comprising two cellulases, CelA and SSO1949 using the 'domain insertion' approach. The SCHEMA tool was used to find the insertion sites on CelA. Figure 14: Crystal structure of Human Pirin (PDB: 1J1L) revealing its Fe 2+ containing site. ..............................................................................................................................................................Figure 15: An example of multicupin: Human homogentisate dioxygenase (HGO). ....Figure 16: Schematic representation of the structure of PMI showing a large interdomain between two conserved barrels containing Zinc in the active site located on the C-terminal side................................................................................................................................Figure 17: 3D structure of PMI showing a central β-barrel catalytic domain flanked by helical domain on one side and a N-terminal β-barrel domain on the other side. ......52 Figure 20 : Agarose gel stained with ETBr of the undigested (Lane 3) and digested plasmid (lanes 1 and 2). Lane 3 shows 3 possible plasmid conformations. ..................... Figure 21: Ligation of the insert coding for TEVc/site and the vector linearized plasmid. The insert is complementary to the remaining Nhe1 fragments of both vector strands..........................................................................................................................................Figure 22: Fragment of the sequence encoding for His6-PMI and comprising the inserted TEVc/site with the insert specific primer (violet frame, primer mix 1) that was used for bacterial colony-screening. ..............................................................................................Figure 23: Agarose gel electrophoresis of PCR products obtained using the positive control primers (primer mix 2) which are complementary to the fragments of the sequence encoding for ampicillin resistance. Lane 1: DNA Ladder .................................... Figure 24: Agarose gel electrophoresis of PCR products obtained using the primer mix 1 which is complementary to fragments of the sequence of the insert i.e. TEVc/site and the vector. Lane 1: DNA Ladder ...............................................................................................Figure 25: Coomassie blue stained SDS-PAGE of supernatant fractions and pellets after the expression of the PMI. Lanes 1, 3, 5 and 7: Supernatant fractions; Lanes 2, 4, Figure 29: Pure His6-PMI and tag free PMI specific activity (AS) as function of the volume of enzymes at 25°C in 50 mM MOPS, pH 7.4 in the presence of all substrates (M6P and NADP + ) and auxiliary enzymes (PGI and G6PDH) as indicated. The reported values are initial velocity (slope of the kinetic divided by the extinction coefficient of NADPH). ....................................................................................................................................................Figure 30: Percentage of the PMI activity as function of added EDTA equiv. .................Figure 33: X-band EPR spectrum measured at 10 K of A) Cu(II)-His6-PMI and B) Cu(II)-PMI. ............................................................................................................................................................. Figure 34: TYCHO measurements of natural PMI-Zn in purple; PMI zinc free in orange and Cu-PMI metalloprotein in pink. ............................................................................................... Figure 35: Left: Structure of PMI crystallized with 5PAHz; Inset: zoom on the active site. ..............................................................................................................................................................Figure 36: Swiss model of PLP showing its β barrel catalytic domain surrounded by an α-helixes domain on one side and a C-terminal β barrel domain on the other side. ..Figure 37: A) X-ray crystal structure of the human Pirin protein (pdb: 1J1L). B) Swiss model of the PLP metalloprotein. .................................................................................................... Figure 38: Heatmap of the goodness of the swiss model at every amino acid of the sequence of human Pirin protein. Blue = predicted with high accuracy; red = predicted with low accuracy. .............................................................................................................Figure 39: Representation of the structure of PLP showing the two conserved barrels of -sheets linked by a single -helixes inter-domain and containing an iron atom in the active site located in the barrel at the N-terminal side.................................................... Figure 46: Insertion of the DNA encoding for the TEV protease cleavage site flanked by the His tag and the PLP linearized sequence. .......................................................................Figure 47: Western Blot image confirming the presence of PCR amplicons. Primary antibody: Anti-His6tag antibody; Secondary antibody: Anti-mouse (H+L) antibody labelled with peroxidase. ....................................................................................................................Figure 49: Western Blot image of His6-PLP. Lanes: 1-Protein Ladder; 2-Flow through; 3-Washed fraction; 5-6-7-8-Eluted fractions. ...........................................................................Figure 50: Right, Coomasie blue stained SDS-PAGE electrophoresis of PLP upon cleavage of the His6 tag (on the right) followed by Western Blot image using Anti His primary antibody (on the left). L: Ladder; Lane 1: His6-PLP + TEV; Lane 2: Tag free PLP ......................................................................................................................................................................Figure 51: Fluorescence titration of the binding of Cu(II) to PLP with 10 equiv. EDTA upon excitation at 280 nm. No significant quenching of fluorescence was observed suggesting the remaining of iron cation in the active site of PLP. ......................................Figure 52: TYCHO measurements of 10 µM native PLP-Fe in black and 10 µM PLP iron free in pink. ..............................................................................................................................................Figure 53: TYCHO measurements of 10 µM native PLP in orange and 10 µM PLP iron free upon addition of 10 mM EDTA in purple. ...........................................................................

	Figure 31: Fluorescence titration of the binding of Cu(NO3)2 to zinc free PMI upon excitation at 280 nm. Increase of the signal with increasing concentrations of added Cu 2+ (0 to 55 equiv.). Measurements were performed in 50 mM MOPS, pH = 7.4 using fractions collected after the purification of His6-PLP on an Ni-NTA agarose column. Lane 2: supernatant; Lane 3: Flow through; Lane 4: washing fraction; Lanes 5,6,7 and 8 4.5 µM PMI. Figure 48: SDS-PAGE electrophoresis followed by Coomassie blue staining of the show bands corresponding to the expected molecular weight of pure His6-PLP of
	about 33 kDa;

[START_REF] Yu | Synthetic fusion protein design and applications[END_REF]

.............................................................................................................

Figure 10: Structures showing the conformational changes of CaM in the closed state (pdb: 1CFD) and bound to Ca 2+ (pdb: 1CLL). 45 ............................................................................

Figure 11: Schematic representation of the recombinant TNF -IL-12 protein comprising the IL-12, TNF and scFv human antibody. 48 ......................................................... Figure 12: Characteristic structure of cupin proteins showing two conserved 2-strands domains separated by a less conserved inter-motif region containing another 2--strands and a loop. .......................................................................................................................

Figure 13: Crystal structure of ACCO showing its unique cupin domain and revealing its Fe 2+ containing active site............................................................................................................. Figure 18: View of the metal binding site in PMI (pdb : 1PMI). Interatomic distances are given in Å. .........................................................................................................................................

Figure 19: Crystal structure of His6-PMI coordinated to the inhibitor 5PAHz. ............... 6 and 8: Pellet fractions L: Protein Ladder. ...................................................................................

Figure 26: Coomassie blue stained SDS-PAGE of fractions collected after the purification on an Ni-NTA agarose column of His6-PMI. Lanes 6, 7, 8 ,9, 10, 11 and 12 show bands corresponding to the expected molecular weight of pure His6-PMI (of about 50 kDa); L: Protein Ladder. ....................................................................................................

Figure 27: Coomassie blue stained SDS-PAGE gel of fractions collected after the purification. Lane 1: supernatant; Lane 2: washed fraction; Lanes 3 and 4 show bands with high intensity corresponding to the molecular weight of pure His6-PMI (about 50 kDa); L: Protein Ladder. ........................................................................................................................

Figure 28: Left: Coomassie blue stained SDS-PAGE; Right: Western Blot image. In Both cases lanes: 1-PMI His; 2-PMI-His + AcTEV; 3-Flow through; 4-Eluted fractions. L: Protein Ladder ........................................................................................................................................ ..............................................................................................................................................

Figure 32: Curve fitting the concentration of Cu-PMI derived from the fluorescence values at 340 nm plotted against total added Cu(II) concentration. ..................................

Figure 40: Structure's resemblance of PMI and PLP. Left: Crystalline structure of PMI; Right: Swiss model of the PLP. ..........................................................................................................

Figure 41 : Western Blot image of the coexpression of the His6-PLP protein and the N-terminal 10 amino acids of LacZ protein fused His6-PLP protein. ..................................

Figure 42: The vector linearized plasmid encoding for PLP showing two Shine Dalgarno (SD) sequences. LacZa 10 amino acids were fused to the 33 kDa PLP protein. ......................................................................................................................................................

Figure 43: Primer design for deletion of a portion of LacZa. Forward and reverse primers flank the region to be deleted. .........................................................................................

Figure 44: The vector linearized plasmid of PLP protein after deletion of a portion of LacZa to avoid the co-expression. ...................................................................................................

Figure 45: Westen Blot image of His6-PLP after the deletion using Anti-His antibody. ...................................................................................................................................................................... L: Ladder ......................................................................................................................

Scheme 21: Construction of the expression vector of the Chimera 2.

Scheme 29: Oxidation reaction of ABTS by H2O2. Scheme 30: Oxidation of TMB by H2O2.

Acknowledgments

ACKNOWLEDGMENTS

First and foremost, I would like to express my special appreciation and thanks to my supervisor Dr. Wadih GHATTAS for his warm welcome, his patient guidance, his encouragement and his support. I have been extremely lucky to have a clever and unique supervisor who cared so much for my work. His dynamism, vision, sincerity, motivation and expertise have deeply inspired me. It was a great honor to work under his guidance. I would also like to thank him for his friendship, empathy and great sense of humor.

LIST OF ABBREVIATIONS ABTS

2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid Chapter 4: Chimeric enzymes The PMI and PLP purified fragments were then triple digested by using the restriction enzymes Fse1, Nde1 and BamH1.

In parallel, the plasmid for we previously used for expressing PMI (pRSET-C vector containing PMI sequence flanked by BamH1 and Nde1 restriction sites) was also double digested by using the restriction enzymes Nde1 and BamH1. The digestion reaction of the plasmid was analyzed by electrophoresis alongside a DNA size marker on a 1.2 % agarose gel. After 45 minutes of migration at 70 Volts, the colorant was at the edge of the gel. The electric current was then stopped, and the gel was visualized with an ultraviolet (UV) transilluminator (Biorad). The superior DNA band, that had the desired MW of the vector (pRSET-C), was cutout and purified while the inferior DNA band corresponding to the PMI's sequence was discarded. DNA extraction from agarose gel followed. The purified vector was mixed with an excess of DNA inserts (3 molar equivalents of DNA sequences encoding for PMI and PLP amplified and digested) and a ligase mix, following the providers procedure (scheme 20). The ligation mixture was used to transform E. coli (DH5alfa) strain. This ligation was repeated several times under various conditions, but transformations all failed.

Scheme 20: Assembly of the purified amplified sequences encoding for the PMI and PLP's active sites in the pRSET-C vector.

To solve this problem, we proceeded with the Gibson assembly method that is specifically designed for gene fragment assembly. New primers were thus designed and new PCRs were run.

The desired fragments of the PMI and PLP vectors were linearized and amplified by PCR using primers containing 20 bp overlap between both fragments. PCR products were purified and added to the Gibson Assembly mix. The reaction was carried out under isothermal conditions by three enzymes provided in the mix: a 5' exonuclease that generates long overhangs, a polymerase that fills in the gaps of the annealed single strand regions, and a DNA ligase that seals the nicks of the annealed and filledin gaps (Figure 77). 5' GCG CGG ATC CGG GTA TCA GAA TTA TGA TTG 3' and 5' GCG CCT GCA GTC AAG CAA AGT CCC GAT AAC CC 3'.

The PCR mixture was prepared using the Q5 site directed mutagenesis kit by adding 12.5 μL 2x Master Mix Q5 hot start kit (NEB) containing the polymerase and its buffer, 2.5 μL primers (final concentration 10 µM), 9 μL nuclease free water and 1 μL of the plasmid (10 pg -1 μg). The PCR was carried out in an applied biosystem thermocycler (StepOne Plus). It started with 30 seconds at 98°C for Taq polymerase activation, followed by 25 4,5-minutes cycles including 10 seconds at 98°C for DNA denaturation, 20 seconds at 65°C for hybridization, 2 minutes at 72°C for extension and two minutes at 72 °C for primer final extension.

The presence of the PCR amplicon and the size of the product were determined by electrophoresis alongside a DNA size marker on a precast 1.2% agarose gel.

o For Half PMI :

The sequence was amplified using the Q5 HiFi PCR NEB kit. The DNA sequence of Half PMI was amplified using two primers: P1 : 5' CGC GGA TCC CAT TTT CTG TTT TTC AAC GCT TTC GT 3' and P2 : 5' CGC GGA TCC GAC TCG AGA TCT GCA GCT GGT A 3'.

The PCR was carried out in a applied biosystem thermocycler. It started with 30 seconds at 98°C for Taq polymerase activation, followed by 30 4-minutes and 50-seconds cycles including 10 seconds at 98°C for DNA denaturation, 20 seconds at 66°C for hybridization, 140 seconds at 72°C for extension and two minutes at 72 °C for primer final extension. Then, the PCR product was purified using the Macherey-Nagel kit.

Concentration and purity were assayed on nanodrop spectrophotometer (Thermo Scientific) and the concentration was equal to 124 ng/µl.

8.1 µl of the purified product was then digested by the 1 µl BamH1 restriction enzyme (NEB) in 5 µl CutSmart Buffer and 35.9 µl sterile water. The optimal digestion time was empirically found to be 1 h. Digestion was stopped thermally by leaving the digestion mixture at 60°C for 20 minutes. It was followed by ligation using the instant sticky ligase master mix kit (NEB). 1.5 µl of the purified product (55 ng/µl) was mixed with 5 µl instant ligase mix (NEB) and 3.5 µl sterile water, following the providers (NEB) procedure. The mixture was then incubated 15 minutes in ice.

5 µl of the ligation mixture was added directly to 50 µl competent cells (NEB) in 14-ml BD Falcon polypropylene round bottom tube, mixed by gentle tapping and incubated -

Iron removal:

The removal of iron from the active site of the PLP was performed by incubating 10mL of 10 µM of a solution of the protein (in 50 mM MOPS, 300 mM NaCl buffer pH = 7,6) with 10 3 equiv. EDTA for 6 hours at 4°C with shaking. Then, the protein was washed 3 times through a vivaspin20 by 30 mL of a 50 mM MOPS buffer pH 7.4 and was finally dialyzed against 2 L of 50 mM MOPS buffer 300 mM NaCl, pH 7.6 for 2 days.

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Protein intrinsic fluorescence:

The fluorescence of the proteins was measured on a FluoroMax-4 spectrofluorometer (Excitation : 280 nm -Emission: 300 nm to 500 nm).

To prepare the artificial enzyme derived from PMI, up to 50 equiv. of Cu (NO3)2 ITC measurements were performed on Microcal Peaq-IT apparatus (Malvern).

To determine the thermodynamic parameters for the binding of metal cations to the proteins, up to 12 equiv. of a solution of metal salt 360 µM in 50 mM MOPS buffer, 300 mM NaCl, pH 7.6 were added to 200 L of a 30 µM solution of PLP in 50 mM MOPS buffer, 300 mM NaCl, pH 7.6. The interaction between PMI and the metal complex was characterized by measuring the heat variation upon the addition of increasing amounts of metal salt.

S, H and Kd were calculated from the fitting of the experimental data.

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TYCHO :

To examine the protein's structural integrity and to determine its folding, measurements were done on Tycho NT.6 (NanoTemper). Changes in intrinsic fluorescence at 350 nm and 330 nm, due to tryptophan and tyrosine residues in the protein were assessed while a heat ramp was applied. Transitions in the folding state of a protein were indicated by changes in the fluorescence signal.

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Peroxidase activity:

The oxidation of ABTS and TMB (scheme 29 and scheme 30), was followed by monitoring the absorbance respectively at 414 and 650 nm of the corresponding products obtained after the oxidation by H2O2 catalyzed by the artificial copper(II)-PMI metalloenzyme.

The