Christiane Luley-Goedl

Bernd Nidetzky
email: bernd.nidetzky@tugraz.at

Carbohydrate synthesis by disaccharide phosphorylases: reactions, catalytic mechanisms and application in the glycosciences

Keywords: Glycosyl transfer, Glycobiotechnology, Enzyme engineering glycosyl transfer, inverting and retaining mechanism, enzyme engineering, glycoside synthesis, application

Disaccharide phosphorylases are glycosyltransferases (EC 2.4.1.-) of specialized carbohydrate metabolism in microorganisms. They catalyze glycosyl transfer to phosphate using a disaccharide as donor substrate. Phosphorylases for the conversion of naturally abundant disaccharides including sucrose, maltose, α,α-trehalose, cellobiose, chitobiose, and laminaribiose have been described.

Structurally, these disaccharide phosphorylases are often closely related to glycoside hydrolases and transglycosidases. Mechanistically, they are categorized according the stereochemical course of the reaction catalyzed, whereby the anomeric configuration of the disaccharide donor substrate may be retained or inverted in the sugar 1-phosphate product. Glycosyl transfer with inversion is thought to occur through a single displacement-like catalytic mechanism, exemplified by the reaction coordinate of cellobiose/chitobiose phosphorylase. Reaction via configurational retention takes place through the double displacement-like mechanism employed by sucrose phosphorylase. Retaining α,α-trehalose phosphorylase (from fungi) utilizes a different catalytic strategy, perhaps best described by a direct displacement mechanism, to achieve stereochemical control in an overall retentive transformation.

Disaccharide phosphorylases have recently attracted renewed interest as catalysts for synthesis of glycosides to be applied as food additives and cosmetic ingredients. Relevant examples are lacto-Nbiose and glucosylglycerol whose enzymatic production was achieved on multikilogram scale. Protein engineering of phosphorylases is currently pursued in different laboratories with the aim of broadening the donor and acceptor substrate specificities of naturally existing enzyme forms, to eventually generate a toolbox of new catalysts for glycoside synthesis.

Introduction

The O-glycosidic linkage is a highly stable bond in many natural molecules. Its formation as in disaccharides, polysaccharides, glycoconjugates and numerous other small molecules is usually catalyzed by glycosyltransferases (Scheme 1A). The activated donor substrate required for enzymatic glycosyl transfer is provided from central metabolic pathways of the cell. Glycosyltransferases are often very specific for both the donor and acceptor substrate used in their reactions [START_REF] Lairson | Glycosyltransferases: structures, functions, and mechanisms[END_REF]. The free energy of conversion of the activated sugar donor drives the glycosyltransferase reaction towards the O-glycosidic product. Breakdown of the O-glycosidic linkage is a task usually fulfilled by glycoside hydrolases (Scheme 1B). In aqueous solution, the thermodynamic equilibrium of the hydrolase reaction lies far on the side of products. The rate acceleration achieved by these hydrolases in comparison to the corresponding uncatalyzed hydrolysis reaction is impressive, sometimes reaching values on the order of 10 17 [2]. Metabolic incorporation of the hydrolysis products usually requires energy, often in the form of ATP. For example, the glucose produced from sucrose (α-Dglucopyranosyl-1,2-β-D-fructofuranoside) would often be converted to glucose 6-phosphate in a kinase-catalyzed transformation. The expenditure of cellular energy in the phosphorylation of sugars derived from hydrolysis of O-glycosidic substrates is a possible disadvantage, especially under conditions in which saccharide reserves are mobilized or in the case of organisms that dwell in a lowoxygen or anoxic environment where ATP production cannot occur via the efficient process of respiratory chain-linked phosphorylation. Phosphorolysis of substrate, that is breakdown of the Oglycosidic linkage concomitant with glycosyl transfer to phosphate, yields the corresponding sugar 1phosphate as a high-energy product [START_REF] Lairson | Mechanistic analogies amongst carbohydrate modifying enzymes[END_REF]. Because no ATP is required in further metabolism of the resulting sugar 1-phosphate, phosphorolysis seems to be an energetically more economic way of utilizing saccharide substrates than is hydrolysis. In agreement with the notion of the physiological advantages resulting from phosphorolysis, enzymes catalyzing phosphorolytic conversion of Oglycosidic substrates have often been identified in microorganism having a facultatively anaerobic lifestyle. Notable examples include Bifidobacterium sp., Leuconostoc sp., Lactobacillus sp., Cellulomonas sp., Cellvibrio sp., and Clostridium sp. Furthermore, intracellular degradation of α-1,4glucan polymers (glycogen, starch) stockpiled as reserves of carbon and energy occurs in a wide range Using the example of phosphorolysis of sucrose, Scheme 1C shows the basic reaction catalyzed by Oglycoside phosphorylases, henceforth also in brief, phosphorylases.

The known phosphorylases utilize disaccharides, oligosaccharides (maltodextrins, cellodextrins) or polysaccharides (starch, glycogen) as donor substrates. Herein, we restrict our discussion mainly to disaccharide phosphorylases for two reasons. First, the disaccharide phosphorylases embrace essentially the complete mechanistic diversity of this group of glycosyltransferases. Second, applications of phosphorylase enzymes in glycoside synthesis were developed chiefly using disaccharide phosphorylases and considerations of enzymatic synthesis and enzyme engineering are a central theme of this article. The family of phosphorylases, which irrespective of their divergent substrate preference for maltodextrins, starch or glycogen, are often collectively called "glycogen phosphorylases", is not covered in detail. However, mechanistic and synthetic analogies between trehalose, sucrose and glycogen phosphorylase are drawn and will be pointed out later.

Disaccharide phosphorylases: classification, structure and function

Phosphorylases bridge in many ways the classes of glycosyltransferases and glycoside hydrolases.

Although categorized by EC number as transferases, phosphorylases have various properties in common with glycoside hydrolases. Unlike "true" glycosyltransferases whose physiological function is synthesis of O-glycosides, phosphorylases are generally believed to fulfil a catabolic role. The intracellular concentration of phosphate may be in the range <5 mM [START_REF] Hollander | Phosphorus-31 nuclear magnetic resonance studies of the effect of oxygen upon glycolysis in yeast[END_REF] and is expected to exceed the concentration of sugar 1-phosphate (e.g. α-D-glucose 1-phosphate) by up to two orders of magnitude [START_REF] Aiello | Intracellular glucose 1-phosphate and glucose 6-phosphate levels modulate Ca 2+ homeostasis in Saccharomyces cerevisiae[END_REF]. The concentration of free acceptor substrate (e.g. D-glucose, D-fructose, N-acetyl-D-glucosamine) in the cell will also be very low [START_REF] Teusink | Intracellular glucose concentration in derepressed yeast cells consuming glucose is high enough to reduce the glucose transport rate by 50%[END_REF]. According to the law of mass action, therefore, the direction of the phosphorylase reaction in which O-glycoside is produced (synthesis) will be strongly disfavoured as compared to phosphorolysis under conditions prevailing in vivo.

According to a widely used sequence-based classification [START_REF] Cantarel | The Carbohydrate-Active EnZymes database (CAZy): An expert resource for Glycogenomics[END_REF], all known disaccharide phosphorylases except retaining trehalose phosphorylase are placed in one of the families of glycoside shown to consist of a partially broken TIM (triosephosphate isomerase) barrel fold [START_REF] Hidaka | The crystal structure of galacto-N-biose/lacto-N-biose I phosphorylase: A large deformation of a TIM barrel scaffold[END_REF] that is structurally similar to a thermophilic β-galactosidase, thus supporting the classification of this and related phosphorylase as one of the current GH families. Membership of phosphorylases to glycoside hydrolase families and clans strongly supports an evolutionary relationship between these two enzyme classes at the level of both structure and catalytic function. Interestingly, therefore, the retaining trehalose phosphorylases and glycogen phosphorylases are classified in glycosyltransferase family GT-4 and GT-35, respectively. It is known from crystal structures (glycogen phosphorylase) and suggested by structure modelling (trehalose phosphorylase) that both phosphorylases adopt the socalled GT-B fold, which comprises two Rossmann fold-like domains. The classification of glycosyltransferases currently spans 92 families. Membership to GT families for glycogen phosphorylase and trehalose phosphorylase would suggest that these two phosphorylases are mechanistically distinct from counterpart phosphorylases categorized in GH families. The comparison between trehalose phosphorylase (family GT-4) and sucrose phosphorylase (family GH-13) has shown that the two enzymes utilize strikingly different reaction coordinates to achieve catalytic efficiency and stereochemical control in their highly analogous substrate transformations [START_REF] Goedl | Mechanistic differences among retaining disaccharide phosphorylases: Insights from kinetic analysis of active site mutants of sucrose phosphorylase and α,α-trehalose phosphorylase[END_REF], as will be described later. Family GT-35 comprises no enzyme activity except that of glycogen phosphorylase. Like glycoside hydrolases and glycosyltransferases, phosphorylases can be categorized according to whether in the enzymatic phosphorolysis reaction the anomeric configuration of the donor substrate is retained or inverted in the corresponding sugar 1-phosphate product [START_REF] Lairson | Mechanistic analogies amongst carbohydrate modifying enzymes[END_REF]. Reaction via inversion and retention are thought to necessitate different catalytic mechanisms. As shown in Table 1, sucrose phosphorylase and trehalose phosphorylase catalyze an α-retentive reaction, involving an overall axial-to-axial substitution on the anomeric carbon of the glycosyl ring [START_REF] Goedl | Mechanistic differences among retaining disaccharide phosphorylases: Insights from kinetic analysis of active site mutants of sucrose phosphorylase and α,α-trehalose phosphorylase[END_REF].

Phosphorylase reaction coordinates featuring the opposite stereochemical course, that is β-retentive conversion via an equatorial-to-equatorial substitution, have not so far been described. Inverting phosphorylases promote conversion of their substrates via either axial-to-equatorial or equatorial-toaxial substitution on the anomeric carbon, as seen in maltose phosphorylase [START_REF] Tsumuraya | Substrate-induced activation of maltose phosphorylase: Interaction with the anomeric hydroxyl group of α-maltose and α-Dglucose controls the enzyme's glycosyltransferase activity[END_REF] and cellobiose phosphorylase [START_REF] Nidetzky | Role of non-covalent enzyme-substrate interactions in the reaction catalyzed by cellobiose phosphorylase from Cellulomonas uda[END_REF], respectively. Chemically, the difference between a phosphorylase reaction and the corresponding hydrolase reaction utilizing the same disaccharide substrate is the choice of nucleophile, phosphate or water. Crystal structures of different phosphorylases reveal how phosphate is accommodated in the enzyme catalytic site and provide suggestions of how phosphorylases might discriminate against reaction with water, preventing "error hydrolysis" of their donor substrate [START_REF] Egloff | Crystal structure of maltose phosphorylase from Lactobacillus brevis: Unexpected evolutionary relationship with glucoamylases[END_REF][START_REF] Hidaka | Structural dissection of the reaction mechanism of cellobiose phosphorylase[END_REF]. With the exception of sucrose phosphorylase that shows significant inherent hydrolase activity [START_REF] Mieyal | Mechanism of action of sucrose phosphorylase. 3. The reaction with water and other alcohols[END_REF], other phosphorylases typically do not show detectable conversion of disaccharide substrate (or an analogue thereof) in a reaction with water as nucleophile [START_REF] Tsumuraya | Substrate-induced activation of maltose phosphorylase: Interaction with the anomeric hydroxyl group of α-maltose and α-Dglucose controls the enzyme's glycosyltransferase activity[END_REF][START_REF] Nidetzky | Role of non-covalent enzyme-substrate interactions in the reaction catalyzed by cellobiose phosphorylase from Cellulomonas uda[END_REF][START_REF] Goedl | Structure-function relationships for Schizophyllum commune trehalose phosphorylase and their implications for the catalytic mechanism of family GT-4 glycosyltransferases[END_REF][START_REF] Schwarz | Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild-type and His334→Gly mutant enzymes[END_REF].

Phosphorylase mechanisms

Cellobiose and chitobiose phosphorylase. The structure and function of these enzymes from family GH-94 have been examined in considerable detail. Crystal structures of cellobiose phosphorylase from Cellvibrio gilvus [17] and chitobiose phosphorylase from Vibrio proteolyticus [START_REF] Hidaka | Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (α/α) 6 barrel fold[END_REF] have been reported.

At the level of the three-dimensional structure (overall fold), cellobiose and chitobiose phosphorylase are very similar one to another, and both enzymes are structurally homologous to maltose phosphorylase from Lactobacillus brevis (family GH-65) [START_REF] Egloff | Crystal structure of maltose phosphorylase from Lactobacillus brevis: Unexpected evolutionary relationship with glucoamylases[END_REF] and glucoamylase from Thermoanaerobacterium thermosaccharolyticum (family GH-15, clan GH-L) [START_REF] Aleshin | Crystal structure and evolution of a prokaryotic glucoamylase[END_REF]. Another cellobiose phosphorylase from Cellulomonas uda has been successfully crystallized [START_REF] Van Hoorebeke | Crystallization and Xray diffraction studies of cellobiose phosphorylase from Cellulomonas uda[END_REF].

Structural comparison of chitobiose phosphorylase, maltose phosphorylase and glucoamylase revealed that Asp-492 (amino acid numbering of chitobiose phosphorylase from V. proteolyticus) superposed well with the known and putative general catalytic acid of the reaction catalyzed by the glucoamylase (Glu-438; from T. thermosaccharolyticum) and maltose phosphorylase (Glu-487; from L. brevis), respectively [START_REF] Hidaka | Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (α/α) 6 barrel fold[END_REF]. Asp-492 is part of a highly conserved region in phosphorylases of family GH-94. Substitution of Asp-492 by Asn resulted in complete loss of activity, suggesting that the Asp side of chitobiose phosphorylase serves an analogous Brønsted acid catalytic role as the homologous Glu side chains of glucoamylase and maltose phosphorylase [START_REF] Hidaka | Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (α/α) 6 barrel fold[END_REF]. Interestingly, at the position of the general catalytic base on an inverting hydrolase (Scheme 2A), no candidate residue is found in chitobiose phosphorylase and maltose phosphorylase. However, the position of the base catalyst in glucoamylase (Glu-636) is almost precisely adopted by Gln-690, another highly conserved residue amongst GH-94 phosphorylases (Figure 1), suggesting that activation of the phosphate ion is not necessary in the reaction as it is for the attack of a water molecule by an inverting hydrolase.

The evidence from crystal structure determination together with results of biochemical studies on chitobiose/cellobiose phosphorylase supports the mechanism summarized in Scheme 2B. Note: despite the differences in stereochemical features of the disaccharide substrate, the basic catalytic mechanism of phosphorolysis with inversion of configuration appears to be conserved among cellobiose phosphorylase [START_REF] Hidaka | Structural dissection of the reaction mechanism of cellobiose phosphorylase[END_REF] and maltose phosphorylase [START_REF] Egloff | Crystal structure of maltose phosphorylase from Lactobacillus brevis: Unexpected evolutionary relationship with glucoamylases[END_REF]. Inverting α,α-trehalose phosphorylase and kojibiose phosphorylase for which no crystal structure has so far been published seem to use a similar direct displacement-like mechanism.

The thorough characterization of chitobiose/cellobiose phosphorylase has provided a wealth of structure-function relationship data that are conducive to the design of novel phosphorylases for carbohydrate synthesis (see later).

Sucrose phosphorylase. Crystal structures of sucrose phosphorylase from Bifidobacterium adolescentis together with evidence from site-directed mutagenesis and other biochemical studies have revealed that this enzyme utilizes a glycoside hydrolase-like double displacement mechanism, The proposed mechanism (Scheme 3), which is strongly supported by the crystallographic data shown in Figure 2, received further support from mutational analyses carried out on the enzyme from Leuconostoc mesenteroides. In this phosphorylase, Asp-196 which is homologous to Asp-192 of the B. adolescentis enzyme was replaced by Ala (D196A) [START_REF] Schwarz | Asp-196→Ala mutant of Leuconostoc mesenteroides sucrose phosphorylase exhibits altered stereochemical course and kinetic mechanism of glucosyl transfer to and from phosphate[END_REF]. In agreement with the expected kinetic consequences resulting from complete removal of catalytic assistance by the nucleophilic group on the enzyme, the purified D196A mutant lacked activity (≤ 0.0001% that of the wild-type enzyme).

However, when presented with azide as an alternative nucleophile, the conversion of α-D-glucose 1phosphate proceeded at a slow rate and resulted in the formation of a new substitution product, glucose 1-azide with a β-anomeric configuration. Activity enhancement in the D196A mutein was therefore suggested to result from the direct participation of azide in the now inverting, single displacement-like mechanism of glucosyl transfer. Results of steady-state kinetic analysis of glucosyl transfer to phosphate catalyzed by D196A suggested reaction through a ternary enzyme-substrate complex, in stark contrast to the wild-type enzyme whose reaction in two catalytic steps via a covalent intermediate appears to dictate Ping-Pong kinetics [START_REF] Schwarz | Asp-196→Ala mutant of Leuconostoc mesenteroides sucrose phosphorylase exhibits altered stereochemical course and kinetic mechanism of glucosyl transfer to and from phosphate[END_REF][START_REF] Mieyal | Disaccharides Phosphorylases[END_REF].

Glu-237 (homologous to Glu-232) was replaced by Gln, so the carboxamide group of the Gln cannot fulfil the acid-base catalytic function proposed for the carboxylic group of the original Glu (Scheme 3). Detailed kinetic characterization of E237Q showed that catalytic steps involving a poor leaving group or nucleophile were slowed by five orders of magnitude in the mutein as compared to the wild-type enzyme whereas other steps expected to proceed without support from an acid-base catalyst were not strongly affected by the site-directed substitution [START_REF] Schwarz | Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu 237 →Gln mutant enzymes[END_REF]. Results of pH studies suggested a role for Glu-237 in which the carboxylic acid/carboxylate side chain of the Glu alternates between catalytic functions of a general acid and base in glucosylation and deglucosylation halfreactions of sucrose phosphorylase, respectively. It was proposed that the pK a of the Glu-237 side chain cycles between 7.2 in the free enzyme and 5.8 in the glucosyl-enzyme intermediate [START_REF] Schwarz | Acid-base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu 237 →Gln mutant enzymes[END_REF]. Recently, the key catalytic amino acids in L. mesenteroides sucrose phosphorylase (Asp-196, Glu-237) were replaced by the corresponding carboxamide residues, and it was shown that the D196N E237Q double mutein was still capable of promoting slow phosphorolysis [START_REF] Goedl | Sucrose phosphorylase harbouring a redesigned, glycosyltransferase-like active site exhibits retaining glucosyl transfer in the absence of a covalent intermediate[END_REF]. An enzymatic reaction coordinate for α-retaining glucosyl transfer via a covalent intermediate was changed for the first time into one where this intermediate was lacking and configurational retention appeared to be achieved through a direct front-side nucleophilic displacement reaction.

Retaining α α α α,α α α α-trehalose phosphorylase. According to Table 1, retaining α,α-trehalose phosphorylase (family GT-4) must be distinguished clearly from "counterpart" inverting trehalose phosphorylase (family GH-65) [START_REF] Van Hoorebeke | Crystallization and X-ray diffraction studies of inverting trehalose phosphorylase from Thermoanaerobacter sp[END_REF]. No crystal structure of a retaining trehalose phosphorylase has been determined so far. However, the structures of several glycosyltransferases related to the trehalose phosphorylase by common membership to family GT-4 have been solved [START_REF] Vetting | Structural and enzymatic analysis of MshA from Corynebacterium glutamicum: Substrate-assisted catalysis[END_REF][START_REF] Martinez-Fleites | Insights into the synthesis of lipopolysaccharides and antibiotics through structures of two retaining glycosyltransferases from family GT-4[END_REF][START_REF] Guerin | Molecular recognition and interfacial catalysis by the essential phosphatidylinositol mannosyltransferase PimA from mycobacteria[END_REF][START_REF] Chua | The structure of sucrose phosphate synthase from Halothermothrix orenii reveals its mechanism of action and binding mode[END_REF][START_REF] Steiner | Structural basis of substrate binding in WsaF, a rhamnosyltransferase from Geobacillus stearothermophilus[END_REF][START_REF] Ruane | Crystal structure of a family GT4 glycosyltransferase from Bacillus anthracis ORF BA1558[END_REF]. A three-dimensional model of trehalose phosphorylase from Schizophyllum commune has thus been created based on this structural basis. In spite of its possible ambiguity, the model is clear in showing that the active-site of the trehalose phosphorylase (Figure 3A) is very different from the active-site of sucrose phosphorylase (Figure 2). However, active-site similarity between trehalose phosphorylase and glycogen phosphorylase is suggested (Figure 3). Mutagenesis data confirm the importance of Lys-512 and Arg-507 as key phosphate-binding residues for the activity of trehalose phosphorylase. [START_REF] Goedl | The phosphate site of trehalose phosphorylase from Schizophyllum commune probed by site-directed mutagenesis and chemical rescue studies[END_REF]. Both trehalose and glycogen phosphorylase lack a suitable amino acid in their respective active-site whose side chain would be positioned appropriately to be a candidate catalytic nucleophile. A direct front-side nucleophilic displacement mechanism was therefore suggested for both enzymes. In literature, this mechanism is sometimes referred to as "internal return-like" (S N i-like) and is considered to be a strong proposal in the current debate about the catalytic mechanism of retaining glycosyltransferases [START_REF] Lairson | Glycosyltransferases: structures, functions, and mechanisms[END_REF].

Biochemical data strongly support the idea that trehalose phosphorylase is mechanistically different from sucrose phosphorylase. Unlike sucrose phosphorylase, which utilizes α-D-glucose 1fluoride as donor substrate for glucosyl transfer to either phosphate or fructose [START_REF] Mieyal | Disaccharides Phosphorylases[END_REF], trehalose phosphorylase recognizes α-D-glucose 1-fluoride exclusively as a slow substrate for the phosphorolysis direction [START_REF] Eis | The stereochemical course of the reaction mechanism of trehalose phosphorylase from Schizophyllum commune[END_REF]. Trehalose phosphorylase does not promote the conversion of α-D- [START_REF] Vandamme | Microbial sucrose phosphorylase: Fermentation process properties, and biotechnical applications[END_REF][START_REF] Kitao | Transglucosylation catalyzed by sucrose phosphorylase from Leuconostoc mesenteroides and production of glucosyl-xylitol[END_REF][START_REF] Van Den Broek | Physicochemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083[END_REF][START_REF] Goedl | Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: Characterization, kinetic studies of transglucosylation, and application of immobilized enzyme for production of α-D-glucose 1phosphate[END_REF][START_REF] Renirie | Regio-and stereoselective glucosylation of diols by sucrose phosphorylase using sucrose or glucose 1-phosphate as glucosyl donor[END_REF]. Nucleophilic competition for reaction with a similar intermediate was not observed in trehalose phosphorylase [START_REF] Eis | Characterization of trehalose phosphorylase from Schizophyllum commune[END_REF].

In summary, therefore, although ambiguity remains regarding the chemical mechanism by which the trehalose phosphorylase functions, it is clear that trehalose phosphorylase is mechanistically distinct from sucrose phosphorylase. Scheme 4 is a plausible suggestion for the mechanism utilized by trehalose phosphorylase. The mechanism employed by glycogen phosphorylase may be similar to that of trehalose phosphorylase. It is interesting that glycogen phosphorylases require a pyridoxal 5'phosphate cofactor for activity [START_REF] Palm | The role of pyridoxal 5'phosphate in glycogen phosphorylase catalysis[END_REF] whereas trehalose phosphorylases are active in the absence of a cofactor. If the role of pyridoxal 5'-phosphate is that of a catalytic acid that promotes (via the substrate phosphate) a partial protonation of the glycosidic oxygen of the donor substrate, there remains the question of how trehalose phosphorylase achieves a similar "activation" of the substrate phosphate in an S N i-like mechanism. Results of pH studies suggested that trehalose phosphorylase recognizes the doubly protonated (monoanionic) form of phosphate [START_REF] Eis | Fungal trehalose phosphorylase: Kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune[END_REF] which may be a sufficiently acidic substrate to facilitate C-O bond cleavage at the level of the ternary complex in the absence of cofactor (Scheme 4).

Novel application of disaccharide phosphorylases in synthesis

Although sugar 1-phosphates are valuable compounds for highly specialized applications, there appears to be limited need for them as fine chemicals. α-D-Glucose 1-phosphate is used in medicine as a substitute of inorganic phosphate in parenteral nutrition and as supplement in the case of a phosphate deficiency [START_REF] Ronchera-Oms | Stability of parenteral nutrition admixtures containing organic phosphates[END_REF]. Therefore, phosphorolysis of a disaccharide substrate is generally of a minor interest in carbohydrate synthesis, except in situations where this conversion is applied to prepare the sugar 1phosphate donor substrate for another phosphorylase-catalyzed transformation, now carried out in synthesis direction (see later). Small-molecule glycosylations performed by phosphorylases are usually highly selective with respect to the site on the acceptor substrate that becomes derivatized. The Oglycosidic products thus obtained typically have a very high regioisomeric purity. The absence of requirement to perform a separation of product isoforms facilitates the downstream processing and contributes substantially to a lowering of the overall production costs.

Inverting phosphorylases are applied under conditions of equilibrium-controlled synthesis.

Donor and acceptor substrates are usually incubated in the presence of enzyme until the conversion is complete. The thermodynamic equilibrium constant (K eq ) of the reaction, disaccharide + phosphate ↔ sugar 1-phosphate + monosaccharide, normally favours synthesis of disaccharide from sugar 1phosphate and acceptor. Note: synthesis of sucrose is an exception (see later)! The K eq value may be dependent on pH and typically, synthesis becomes more preferred as the pH is decreased. The molar ratio of donor and acceptor can be varied to alter the composition of the product mixture at the end of the conversion. Considerations of product work-up often dictate the choice of these reaction conditions. With some phosphorylases, sugar 1-fluorides are interesting alternate donor substrates that replace the sugar 1-phosphate in the synthesis reaction. Cellobiose phosphorylase and maltose phosphorylase [START_REF] Tsumuraya | Substrate-induced activation of maltose phosphorylase: Interaction with the anomeric hydroxyl group of α-maltose and α-Dglucose controls the enzyme's glycosyltransferase activity[END_REF] are relevant examples. The overall conversion, sugar 1-fluoride + monosaccharide → disaccharide + fluoride -+ H + , is essentially irreversible. It was shown for synthesis of cellobiose from α-D-glucose 1-fluoride and glucose catalyzed by cellobiose phosphorylase that quantitative yields of disaccharide product can be obtained [START_REF] Nidetzky | Cellobiose phosphorylase from Cellulomonas uda: Gene cloning and expression in Escherichia coli, and application of the recombinant enzyme in a 'glycosynthase-type' reaction[END_REF]. The corresponding yield in the analogous transformation, in which α-D-glucose 1-phosphate was used instead of α-D-glucose 1-fluoride, was substantially lower. Fluoride does not serve as nucleophilic acceptor in the conversion of cellobiose by cellobiose phosphorylase to a measurable extent. Because the phosphorylase cannot hydrolyze the cellobiose formed, the disaccharide product is stable. By analogy to the "glycosynthase technology", which is based on mutated glycoside hydrolases that utilize activated glycosyl donors like sugar 1- 12 fluorides for glycoside synthesis but are unable to hydrolytically degrade the resulting products [START_REF] Perugino | Oligosaccharide synthesis by glycosynthases[END_REF][START_REF] Rakic | Recent developments in glycoside synthesis with glycosynthases and thioglycoligases[END_REF], these authors previously used the term "glycosynthase-like" to described the synthetic reaction of a wild-type phosphorylase with a sugar 1-fluoride as donor substrate [START_REF] Nidetzky | Cellobiose phosphorylase from Cellulomonas uda: Gene cloning and expression in Escherichia coli, and application of the recombinant enzyme in a 'glycosynthase-type' reaction[END_REF]. Nakai et al. recently showed utilization of α-D-glucose 1-fluoride as donor substrate for reaction catalyzed by cellobiose and cellodextrin phosphorylases from Clostridium thermocellum [START_REF] Nakai | Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum[END_REF].

The donor substrate specificity of the known inverting phosphorylases is narrow. Small structural modifications of the substrate like epimerisation or deoxygenation at a single carbon atom usually result in large losses of transferase activity. Application of wild-type phosphorylases in glycosylation reactions using non-natural glycosyl donors is therefore often not practically useful. The replacement of sugar 1-phosphate by the corresponding sugar 1-fluoride may be an exception. The scope of acceptor substrates is, however, often broad. Cellobiose phosphorylase has been used for glucosylation of a variety of acceptors, including disaccharides [START_REF] Percy | Acceptor specificity of cellobiose phosphorylase from Cellvibrio gilvus: Synthesis of three branched trisaccharides[END_REF][START_REF] Rajashekhara | Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium, Thermotoga maritima MSB8[END_REF][START_REF] Kino | A new method of synthesis of alkyl β-glycosides using sucrose as sugar donor[END_REF] and the same applies to maltose phosphorylase [START_REF] Kino | Enzymatic synthesis of αanomer-selective D-glucosides using maltose phosphorylase[END_REF][START_REF] Nakai | The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel α-glucosides through reverse phosphorolysis by maltose phosphorylase[END_REF], inverting trehalose phosphorylase [START_REF] Maruta | Acceptor specificity of trehalose phosphorylase from Thermoanaerobacter brockii: production of novel nonreducing trisaccharide, 6-O-alpha-D-galactopyranosyl trehalose[END_REF] and kojibiose phosphorylase [START_REF] Chaen | Enzymatic synthesis of novel oligosaccharides from L-sorbose, maltose, and sucrose using kojibiose phosphorylase[END_REF].

Depending on the reaction mechanism utilized, retaining phosphorylases offer the possibility to perform glycoside synthesis under thermodynamic or kinetic control. Phosphorylases operating by a ternary complex kinetic mechanism in which both substrates must bind to the enzyme before a product can be released need to be applied under equilibrium-controlled reaction conditions, as described above for the inverting phosphorylases. Trehalose phosphorylase and glycogen phosphorylase belong to this group. The scope of donor substrates utilized by these two enzymes is relatively small. While there have been reports on transfer of mannosyl [START_REF] Evers | Further syntheses employing phosphorylase[END_REF] and 2-deoxy-glucosyl residues [START_REF] Klein | General acid-base catalysis of α-glucan phosphorylase: Stereospecific glucosyl transfer from D-glucal is a pyridoxal 5'-phosphate and orthophosphate (arsenate) dependent reaction[END_REF] by glycogen phosphorylase, the activity of this enzyme for reaction with donor substrates other than glucosyl (α-Dglucose 1-phosphate) is extremely small and hardly sufficient for synthesis. α-D-Glucose 1-fluoride is a slow substrate of trehalose phosphorylase that is only utilized in phosphorolysis direction [START_REF] Eis | The stereochemical course of the reaction mechanism of trehalose phosphorylase from Schizophyllum commune[END_REF].

Conversion of α-D-glucose 1-fluoride by glycogen phosphorylase requires the presence of catalytic amounts of phosphate [START_REF] Palm | α-Glucan phosphorylases catalyze the glucosyl transfer from α-D-glucosyl flouride to oligosaccharides[END_REF]. However, as in cellobiose phosphorylase, for example, the acceptor substrate specificity of trehalose phosphorylase is more relaxed than the corresponding donor substrate specificity [START_REF] Nidetzky | α-Retaining glucosyl transfer catalyzed by trehalose phosphorylase from Schizophyllum commune: Mechanistic evidence obtained from steadystate kinetic studies with substrate analogues and inhibitors[END_REF]. which is distinct from the direct glycosylation catalyzed by phosphorylases that operate by a ternary complex mechanism. Glucosylated sucrose phosphorylase (β-Glucosyl-SPase) can be produced using sucrose, α-D-glucose 1-phosphate or α-D-glucose 1-fluoride as donor substrate [START_REF] Goedl | Sucrose phosphorylase: A powerful transglucosylation catalyst for synthesis of α-D-glucosides as industrial fine chemicals[END_REF]. Sucrose is often preferred for its high stability in the absence of phosphorylase, the high internal energy that drives the reaction, and the good availability at low costs. In the absence of phosphate that would otherwise react with the intermediate, external acceptors (e.g. glycerol) compete with water for participating as nucleophiles in the overall conversion (Scheme 5). Using sucrose, the synthetic reaction catalyzed by the enzyme is therefore, sucrose + glycerol ↔ α-D-glucosyl glycerol + D-fructose. Hydrolysis of the β-glucosyl enzyme intermediate occurs as a side reaction in this process and yields D-glucose. An efficient synthesis must therefore strive for preventing loss of donor substrate to the hydrolysis path (see later). Often this can be achieved by using a suitable excess of acceptor substrate [START_REF] Goedl | A high-yielding biocatalytic process for the production of 2-O-(α-D-glucopyranosyl)-sn-glycerol, a natural osmolyte and useful moisturizing ingredient[END_REF]. In sucrose phosphorylase, however, use of high sucrose concentration have also been found to be effective in suppressing donor substrate hydrolysis [START_REF] Sawangwan | Single-step enzymatic synthesis of (R)-2-O-α-D-glucopyranosyl glycerate, a compatible solute from micro-organisms that functions as a protein stabiliser[END_REF][START_REF] Luley-Goedl | Regioselective Oglucosylation by sucrose phosphorylase: A promising route for functional diversification of a range of 1,2-propanediols[END_REF]. Because α-D-glucosyl glycerol and other glucosylated acceptors produced by sucrose phosphorylase are by definition substrates for degradation by the enzyme via secondary hydrolysis (or phosphorolysis), their synthesis takes place under kinetic control.

Depending on how fast the respective glucosylated acceptor is broken down, the time of incubation may be a critical parameter in the optimization of the transglycosylation.

Multi-step enzymatic synthesis of disaccharides and other glycosides.

Coupling to sucrose conversion. Scheme 6 shows how phosphorolysis of sucrose is utilized to prepare the α-D-glucose 1-phosphate for a coupled enzymatic reaction catalyzed by another phosphorylase.

The K eq for the conversion of sucrose and phosphate into α-D-glucose 1-phosphate and D-fructose is around 44 at pH 7.0 and 30 °C [START_REF] Goedl | Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: Characterization, kinetic studies of transglucosylation, and application of immobilized enzyme for production of α-D-glucose 1phosphate[END_REF]. Using the combined action of sucrose and glucan phosphorylase, the synthesis of pure amylose for the use as a functional biomaterial could be implemented in high yield [START_REF] Waldmann | The enzymic utilization of sucrose in the synthesis of amylose and derivatives of amylose, using phosphorylases[END_REF][START_REF] Kitamura | Biodegradable articles obtained from enzymatically synthesized amylose[END_REF]. Additional isomerization of D-fructose into D-glucose provides the acceptor substrate for synthesis of various "glucobioses", including α,α-trehalose [START_REF] Schwarz | Trehalose phosphorylase from Pleurotus ostreatus: Characterization and stabilization by covalent modification, and application for the synthesis of α,α-trehalose[END_REF][START_REF] Saito | Purification and characterization of a trehalose synthase from the basidiomycete Grifola frondosa[END_REF] and cellobiose [START_REF] Taniguchi | Method for preparing cellobiose[END_REF]. Other 14 types of phosphorylase have also been coupled as described in literature [START_REF] Aisaka | Enzymatic synthesis of novel disaccharides using disaccharide phosphorylases[END_REF][START_REF] Yamamoto | Hyper expression of kojibiose phosphorylase gene and trehalose phosphorylase gene from Thermoanaerobacter brockii ATCC35047 in Bacillus subtilis and selaginose synthesis utilizing two phosphorylases[END_REF][START_REF] Yoshida | Production of trehalose from starch by maltose phosphorylase and trehalose phosphorylase from a strain of Plesiomonas[END_REF][START_REF] Murao | Enzymic synthesis of trehalose from maltose[END_REF][START_REF] Kitaoka | Conversion of sucrose into cellobiose using sucrose phosphorylase, xylose isomerase, and cellobiose phosphorylase[END_REF]. However, for thermodynamic reasons, production of the intermediary α-D-glucose 1-phosphate is much less efficient in terms of yield when using cellobiose [START_REF] Ohdan | Enzymatic synthesis of amylose[END_REF][START_REF] Yanase | α-Glucan phosphorylase and its use in carbohydrate engineering[END_REF] and even glycogen or starch as donor substrate [START_REF] Suzuki | Synthesis of cellobiose from starch by the successive actions of two phosphorylases[END_REF], as compared to sucrose. This often prevents one-pot synthesis in the presence of two phosphorylases and requires that α-D-glucose 1-phosphate is prepared first in a separate reaction.

Synthesis of β-D-glucose 1-phosphate from maltose [START_REF] Aisaka | Enzymatic synthesis of novel disaccharides using disaccharide phosphorylases[END_REF][START_REF] Nidetzky | Maltodextrin phosphorylase from Escherichia coli: Production and application for the synthesis of αglucose-1-phosphate[END_REF] and trehalose [START_REF] Van Der Borght | Enzymatic production of β-Dglucose-1-phosphate from trehalose[END_REF] is also lacking efficiency however, because of its relevance as glucosyl donor for both enzymatic and chemical glucosylation reactions, the concept of a coupled one-pot phosphorolysis/reverse phosphorolysis reaction using maltose phosphorylase was examined. The costly intermediary β-D-glucose 1phosphate, obtained from maltose in the presence of only 2-fold excess of phosphate, is directly used as substrate for reverse phosphorolysis with different monosaccharides, resulting in >84% yield of new α-(1→4)-glucosidic disaccharides [START_REF] Nakai | Efficient one-pot enzymatic synthesis of α-(1→4)-glucosidic disaccharides through a coupled reaction catalyzed by Lactobacillus acidophilus NCFM maltose phosphorylase[END_REF]. The product spectrum strongly depends on the enzyme's acceptor substrate specificity.

Lacto-N-biose. This disaccharide (Galβ1→3GlcNAc) is a key structural component of human milk oligosaccharides such as lacto-N-tetraose (Galβ1→3GlcNAcβ1→3Galβ1→4Glc) and lacto-Nfucopentaose (Fucα1→2Galβ1→3GlcNAcβ1→3Galβ1→4Glc). It is widely accepted that human milk oligosaccharides have a "prebiotic effect" in that they can provide selective stimulation to the growth of microorganisms such as Bifidobacteria whose presence in the intestinal flora is considered to be beneficial for human health and well-being. The physiological efficacy of these human milk oligosaccharides appears to be linked to the presence of the lacto-N-tetraose unit [START_REF] Xiao | Distribution of in vitro fermentation ability of lacto-N-biose I, a major building block of human milk oligosaccharides, in bifidobacterial strains[END_REF]. Nishimoto and Kitaoka developed an elegant procedure (Scheme 7) in which four enzymes, among them two phosphorylases, were used in a one-pot transformation of sucrose (660 mM) and GlcNAc (600 mM) to give lacto-N-biose (∼500 mM) and fructose in a yield of 83% [START_REF] Nishimoto | Practical preparation of lacto-N-biose I, a candidate for the bifidus factor in human milk[END_REF]. Phosphate (P i ) and UDP-glucose (UDP-Glc) had to be present in catalytic concentrations. The conversion was performed on 10-L scale and following an easy work-up that included treatment with yeast cells and crystallization, about 1.4 kg of pure product was obtained. Using a similar approach in which GalNAc was used instead of GlcNAc as galactosyl acceptor substrate, the same authors reported synthesis of galacto-N-biose disaccharide and the core 1 sugar chain in mucin glycoproteins [START_REF] Nishimoto | One-pot enzymatic production of β-Dgalactopyranosyl-(1→3)-2-acetamido-2-deoxy-D-galactose (galacto-N-biose) from sucrose and 2-acetamido-2-deoxy-D-galactose (N-acetylgalactosamine)[END_REF]. Synthesis of lacto-N-biose as well as that of galacto-N-biose are beautiful examples of the large potential of multi-step enzymatic transformations in making complex carbohydrates available as fine chemicals. For example, the possible application of lacto-N-biose as functional food additive appears to have been opened up by the biocatalytic process indicated in Scheme 7.

Glucosylglycerol. Glycosylglycerols are powerful osmolytes that are produced by various plants, algae, and bacteria in adaptation to salt stress and drought [START_REF] Hincha | Stabilization of model membranes during drying by compatible solutes involved in the stress tolerance of plants and microorganisms[END_REF]. Among them, 2-O-(α-Dglucopyranosyl)-sn-glycerol (GG, Figure 4) has attracted special attention for its promising application as moisturizing agent in cosmetics [START_REF] Yoshida | α-D-Glucopyranosyl glycerol derivatives as antiallergic agents, health foods, and cosmetics[END_REF][84][START_REF] Thiem | Cosmetic preparations with an effective amount of glycosylglycerides as skin moisturizers[END_REF] but also for use as low-caloric sweetener that does not cause tooth decay [86] and may have a prebiotic effect. Possible therapeutic applications of GG that are inferred from its ability to stabilize proteins and cells are currently evaluated [START_REF] Nitta | Antitumor agents containing α-D-glucopyranosylglycerols and food and cosmetics containing them[END_REF][START_REF] Colombo | Glycoglycerolipid analogues active as anti-tumor-promoters: The influence of the anomeric configuration[END_REF]. However, the development of industrial applications for GG (and related carbohydrate-based compatible solutes) has been severely restricted by compound availability. A new biocatalytic process using sucrose phosphorylase according to Scheme 5 has been described which overcomes the chemical and technological challenges of production of stereochemically pure GG as an industrial chemical [START_REF] Goedl | A high-yielding biocatalytic process for the production of 2-O-(α-D-glucopyranosyl)-sn-glycerol, a natural osmolyte and useful moisturizing ingredient[END_REF].

Under optimized reaction conditions using excess of glycerol (2.0 mol/L) over sucrose (0.8 mol/L), about 90% of the applied donor substrate was converted into GG. Enzymatic production of GG [START_REF] Goedl | Preparation of 2-O-glycerylα-D-glucopyranoside from a glucosyl donor and a glucosyl acceptor[END_REF] was implemented on industrial scale by the German company bitop AG, and GG was commercialized under the tradename Glycoin®. A product termed Glycoin Extremium is available on the market and will be used as active ingredient for cosmetic formulations Cellobiose phosphorylase. Lactose would be an interesting donor substrate for phosphorylasecatalyzed transformations but natural enzymes capable of transferring the galactosyl moiety of lactose efficiently appear to be lacking. Cellobiose phosphorylase (from C. uda), which displays a low level of activity towards lactose in the wild-type form, was used as point of departure for the development of engineered lactose phosphorylases [START_REF] De Groeve | Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase[END_REF]. Directed evolution was combined with site saturation mutagenesis to obtain a doubly mutated cellobiose phosphorylase (Thr-508→Ile and Asn-667→Ala) in which a 7.5-fold increase in specific activity on lactose was observed. With Asn-667 being situated adjacent to the donor binding site and Thr-508 being located on a loop at the active-site entrance these mutations mainly affected steps of catalysis or product release rather than substrate binding. Using permeabilized E. coli cells, the engineered "lactose phosphorylase" was employed in the synthesis of expensive α-D-galactose 1-phosphate from low-cost substrates such as lactose and phosphate, lactose + phosphate → α-D-galactose 1-phosphate + glucose. Highly pure α-D-galactose 1-phosphate (9.5 g) was obtained from a 1-L reaction mixture (500 mM lactose, 50 mM phosphate at 50°C for 48h) after anion exchange chromatography and ethanol precipitation [START_REF] De Groeve | Enzymatic production of α-D-galactose 1-phosphate by lactose phosphorolysis[END_REF].

To assist in the engineering of phosphorylases, De Groeve et al. (2010) developed a novel high-throughput screening assay [START_REF] De Groeve | Development and application of a screening assay for glycoside phosphorylases[END_REF]. This assay is based on colorimetric determination of inorganic phosphate released by phosphorylase-catalyzed glycosyl transfer. Application of the assay was demonstrated in the screening of phosphorylase libraries for both novel donor and acceptor specificities. A Glu-649→Cys variant of cellobiose phosphorylase from C. uda was identified that showed activity towards alkyl-β-glucoside and phenyl-β-glucoside acceptors. A robust highthroughput screening platform is a prerequisite for targeted development of glycoside phosphorylases with altered substrate specificities to be employed in the synthesis of new glycosides. A triple mutant, containing both donor (T508I/N667A) and acceptor (E649C) mutations, was used as starting point for creation of more promiscuous enzymes using semi-rational design and site-saturation mutagenesis.

Two residues near the active-site entrance, Asn-156 and Asn-163 were identified to define specificity towards anomerically substituted acceptors. While the wild-type can not glycosylate these acceptors at all, N156D/N163D/T508I/E649C/N667A showed activity towards alkyl-and aryl-β-glucosides and β- Maltose phosphorylase. By using a multiple sequence alignment (Figure 5) and by careful inspection of the crystal structure of maltose phosphorylase from L. brevis [START_REF] Egloff | Crystal structure of maltose phosphorylase from Lactobacillus brevis: Unexpected evolutionary relationship with glucoamylases[END_REF], Svensson and co-workers identified loop 3 of the (α/α) 6 -barrel catalytic domain as key determinant for regioselectivity in this group of phosphorylases. Relevant regions of loop 3 in maltose phosphorylase from L. acidophilus (His-413 to Glu-421) were substituted by the corresponding segments from bacterial inverting trehalose and kojibiose phosphorylases that are related to maltose phosphorylase by common membership to family GH-65. Phosphorylase variants with superior activity for formation of trehalose (α-1,1) and kojibiose (α-1,2) than formation of maltose (α-1,4) were obtained [START_REF] Nakai | Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase[END_REF].

Sucrose phosphorylase. Using random mutagenesis, eight amino acid substitutions, all located between β-strand 2, 3, and 4 (numbering refers to the structure of B. adolescentis sucrose phosphorylase) were identified to contribute to enhancement of high-temperature stability of sucrose phosphorylase from Streptococcus mutans [START_REF] Fujii | Enhancing the thermal stability of sucrose phosphorylase from Streptococcus mutans by random mutagenesis[END_REF]. The wild-type enzyme was almost completely inactivated when incubated at 60°C for 20 min whereas a combination of those eight mutations gave an enzyme retaining 60% of its initial activity under these conditions (Figure 6). Interestingly, the temperature and pH optimum were still the same as reported for the wild-type enzyme while covalent immobilization of sucrose phosphorylase from B. adolescentis on Sepabeads EC-HFA resulted in a shift of the temperature optimum from 58 to 65°C [START_REF] Cerdobbel | Increasing the thermostability of sucrose phosphorylase by multipoint covalent immobilization[END_REF]. The same phosphorylase was stabilized by formation of cross-linked enzyme aggregates [START_REF] Cerdobbel | Sucrose phosphorylase as cross-linked enzyme aggregate: Improved thermal stability for industrial applications[END_REF]. Immobilized preparations of sucrose phosphorylase (from L. mesenteroides) are common in the literature [START_REF] Goedl | Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: Characterization, kinetic studies of transglucosylation, and application of immobilized enzyme for production of α-D-glucose 1phosphate[END_REF][START_REF] Pimentel | Immobilized sucrose phosphorylase from Leuconostoc mesenteroides[END_REF]

  cell types through the action of a phosphorylase that produces glucose 1-phosphate.

  , there are 118 glycoside hydrolase families and phosphorylases are found in families GH-13, GH-65, GH-94 and GH-112 (Table1). Families GH-13 and GH-65 contain phosphorylases and hydrolases. Families GH-94 and GH-112 so far comprise only phosphorylases. Family GH-65 has been categorized into glycoside hydrolase clan GH-L. Member families of clan GH-L share a common (α/α) 6 protein fold. Although, by the criterion of common fold, family GH-94 would also belong to clan GH-L, family-to-clan assignment has not yet been performed for family GH-94. The anomeric configuration of the glycosides cleaved by enzymes from family GH-65 (α-Dglucosides) and family GH-94 (β-D-glycosides) is different, providing a possible reason for not classifying both families into the same clan. Family GH-13 belongs to clan GH-H, which comprises proteins having a common (β/α) 8 fold. No assignment to a particular GH clan has been made for family GH-112. However, the catalytic domain of galacto-N-biose/lacto-N-biose phosphorylase was

  , family GT-4 contains both glycosyltransferase and phosphorylase activities and represents a variety of donor and acceptor substrate specificities.

  3, to convert its disaccharide substrate and phosphate into α-D-glucose 1phosphate and fructose. Three acidic residues (Asp-192, Glu-232, Asp-290) are contributing to the catalytic sub-site, as shown in Figure2. These residues are highly conserved in members of family GH-13.

13 The

 13 covalent β-glucosyl enzyme intermediate formed in the reaction catalyzed by sucrose phosphorylase is point of departure for another route of synthesis, generally called transglycosylation,

15 (

 15 Galβ1→3GalNAc), which is an important core structure in functional sugar chains such as T-antigen

  [90]. Technical features important for successful development of the enzyme-catalyzed process included the use of cheap raw materials, ability of the donor substrate sucrose to drive the reaction to near completion, and the high efficiency and regioselectivity of sucrose phosphorylase in the glucosylation of glycerol.

  β-cellobiosides whose glycosylated derivatives are of interest as novel surfactants, cellulose inhibitors or prebiotic sugars[START_REF] De Groeve | Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides[END_REF].

Figure and Scheme legendsFigure 1 .

 1 Figure and Scheme legends

Figure 2 .

 2 Figure 2. Close-up structure of the active-site of sucrose phosphorylase from Bifidobacterium

Figure 3 .

 3 Figure 3. Close-up structures of the active-sites of GT-4 trehalose phosphorylase from Schizophyllum

Figure 4 .

 4 Figure 4. Appearance and chemical structure of 2-O-(α-D-glucopyranosyl)-sn-glycerol (GG).

Figure 5 .

 5 Figure 5. Multiple sequence alignment of loop 3, connecting α3 and α4 of the (α/α) 6 -barrel catalytic

Figure 6 .Scheme 1 .

 61 Figure 6. Thermal stability of wild-type and engineered Streptococcus mutans sucrose phosphorylase.

  Scheme 1 ((Please keep schemes in file))

  intermediate is a likely mechanistic requirement for the reversible exchange reaction, α-D-glucose 1-phosphate + arsenate → α-D-glucose 1-arsenate + phosphate, which is catalyzed by sucrose phosphorylase with high specific activity. Note that spontaneous hydrolysis of α-D-glucose 1arsenate makes the overall arsenolysis of α-D-glucose 1-phosphate a completely irreversible process.Lacking catalytic assistance to arsenolysis of α-D-glucose 1-phosphate does obviously not prove the absence of a double displacement-like intermediate for trehalose phosphorylase. However, the result supports the notion that in contrast to sucrose phosphorylase, formation and breakdown of any such intermediate in trehalose phosphorylase cannot be analyzed as kinetically isolated reaction steps. The glucosyl-enzyme intermediate of sucrose phosphorylase can be intercepted by numerous alcohol acceptors other than fructose

	glucosyl-enzyme
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	10

glucose 1-phosphate into Glc and phosphate when arsenate is present. A two-step reaction via a

Wiley-VCH Biotechnology Journal

Conclusions

Disaccharide phosphorylases represent a relatively small group of carbohydrate-active enzymes.

Notwithstanding, they embrace a marked diversity of catalytic mechanisms utilized in breakdown or formation of O-glycosidic bonds that spans the classes of glycoside hydrolases and glycosyltransferases. Phosphorylases, in native or engineered form, are useful catalysts for the synthesis of disaccharides and other O-glycosides. An industrial process for synthesis of GG has been developed using sucrose phosphorylase. The use of phosphorylases as a modular assembly system for generation of a whole new glycosidic assortment might be not that far off in the future.


Reindert Nijland
email: r.nijland@umcutrecht.nl

J Grant

Professor J Grant Burgess
email: grant.burgess@ncl.ac.uk

Bacterial Olfaction 2 3

Keywords: Science Communication Bacillus licheniformis, ammonia, olfaction Bacillus licheniformis, ammonia, olfaction, 12 13

Sensing their environment is a crucial ability of all life forms. In higher 2 eukaryotes the sensing of airborne volatile compounds, or olfaction, is well 3 developed. In plants, slime moulds and yeast there is also compelling evidence 4

that these organisms can smell their environment and respond accordingly. Here 5 we show that bacteria are also capable of olfaction. Bacillus licheniformis was 6 able to sense airborne volatile metabolites produced by neighbouring bacterial 7 cultures and cells could respond to this chemical information in a coordinated 8 way. When Bacillus licheniformis was grown in a microtitre plate adjacent to a 9 bacterial culture of the same or a different species, growing in complex medium, 10 biofilm formation and pigment production were elicited by volatile molecules. A 11 weaker response occurred in increasingly distant wells. The emitted volatile 12 molecule was identified as ammonia. These data demonstrate that B. licheniformis 13 has evolved the ability collect information about its environment from the 14 surrounding air and physiologically respond to it in a manner similar to olfaction. 15 This is the first time that a behavioural response triggered by odorant molecules 16 received through the gas phase is described in bacteria. In higher eukaryotes olfaction is an important sensory mechanism which provides 1 the organism with information about its environment. Olfaction is used to detect 2 the presence of food, predators and also other individuals of the same species. 3

Highly advanced olfactory systems are commonly found in animals, both 4 vertebrates and invertebrates (1). Plants can also detect volatile compounds and 5 respond accordingly (2, 3). Already in more primitive eukaryotes olfactory 6 systems are present, and it has been shown that Dictyostelium (4, 5) and yeast (6-7 8) and can sense and respond to volatile chemical gradients such as ammonia. For 8

Dictyostelium it has been shown that the organism can produce ammonia 9 gradients and respond to such gradients by slug formation and migration (4, 5). 10 Separate yeast colonies communicate using ammonia pulses (6, 7), which results 11 in growth inhibition of the part of the colonies facing each other. 12 Cell-cell communication in bacteria is generally achieved using small 13 molecules, such as peptide pheromones, acylated homoserine lactones and other 14 low molecular weight natural products (9-12). Cell-cell communication has been 15 studied in bacteria which are in direct contact in biofilms, or most commonly, in 16 the liquid phase (9, 10). Bacteria are also known to produce a wide variety of 17 volatile metabolites and other chemicals including ammonia (13) through 18 overflow metabolism [START_REF] Russell | Energetics of bacterial growth: balance of 1 anabolic and catabolic reactions[END_REF]. Although bacteria are known to engage in chemical 19 communication with plants (15) and fungi (16) via gaseous molecules, to date 20 there have been no reports of intra-bacterial information exchange through the gas 21 phase. For bacteria the presence of a sensory system that detects volatile 22 compounds and which will inform bacteria of the presence of nearby competitors 1 or food sources would be highly advantageous. 2 3

We have been studying laboratory strains and marine isolates of Bacillus 4 licheniformis as a model system for understanding growth, communication and 5 competition within biofilms (17)(18)[START_REF] Nijland | Transformation of 15 environmental Bacillus subtilis isolates by transiently inducing genetic 16 competence[END_REF]. We grew Bacillus licheniformis DSM13 for 6 2 days in complex medium (LB broth, Oxoid, UK) or biofilm specific medium 7 (MSgg-medium (20)) in clear 96 well flat bottom polystyrene tissue culture 8 microtitre plates (BD-Falcon, USA) with 200µl of culture per well with a closed 9 lid in a plastic container at 30ºC. When growing B. licheniformis in this setup 10 using different growth media in different wells, we observed an unexpected 11 induction of biofilm formation and pigment production. This effect was 12 specifically observed in wells containing B. licheniformis in the biofilm 13 promoting medium MSgg (20), which were adjacent but physically distant to B. 14 licheniformis cultures growing in LB medium (Fig. 1). In wells directly next to 15 the inducer cultures, dense pellicle formation and pigment production was 16 elicited. A weaker response was observed in increasingly distant wells (Fig. 1, 17

Fig. 2). 18

To identify the signal that caused this pigmented pellicle (biofilm) 19 formation, biofilm induction experiments were performed with different growth 20 media and bacterial strains as the inducing cultures. Bacillus subtilis, B. 21 licheniformis, Micrococcus luteus and Escherichia coli were tested as inducers, 22 and all strains were able to induce a similar response in B. licheniformis grown in 23 MSgg medium. Generally, a response was observed when culture medium was 1 complex and nutrient rich, or when a defined medium containing ammonium 2 sulphate was used for growth of the emitting culture. No induction was observed 3 if the sole nitrogen source in the inducing culture medium was glutamate. Two 4 day old, stationary phase cultures and even sterile spent supernatant of nutrient 5 rich cultures elicited the effect. Furthermore, after autoclaving the spent 6 supernatant the induction was still observed. Lowering the pH of the inducing 7 culture from pH 8 to pH 5 abolished the inducing effect (Fig 2A .), and increasing 8 the pH to 9 increased the inducing effect (not shown). These observations 9 indicated that the inducing compound was ammonia. 10 11 We confirmed that ammonia was present in all inducing cultures using Nesslers 12 reagent (21). The presence of ammonia is not surprising, since bacterial 13 metabolism is notoriously leaky (22) and when growing under high nutrient 14 conditions many bacteria (13), including B. licheniformis (23), produce ammonia. 15

The ammonia present in the aqueous solution is in equilibrium with ammonium 16 ions (NH 4 + ), depending on the pH of the solution. Since the NH 4 + ion will not 17 easily escape from solution, the airborne volatile NH 3 molecules are responsible 18 for the observed effect. The ammonia dissolves on contact with the cells in 19 suspension in the wells and an NH 3 -NH 4 + equilibrium will be established. 20

21

The inducing effect of ammonia was subsequently confirmed by using a solution 22 of ammonia to test for induction. We used the same microtitre plate setup, as 23 Optima plate reader (BMG labtech, UK). The cultures were flanked by one 3 column of wells containing an aqueous solution of ammonia at 25mM (left) and 4 2.5mM (right). Absorbance was measured at OD 595 every 30 minutes and the 5 average growth curve of 8 replicate wells was plotted. A 0.05% ammonia solution 6 in H 2 O (25mM) showed a similar induction as observed previously with the 7 inducing cultures (Fig. 2B). 8

The nitrogen source in the recipient culture in MSgg medium was 34mM 9 glutamate. The use of an ammonium containing nitrogen source in the growth 10 medium would also lead to the release of volatile NH 3 into the gas phase. This 11 makes it difficult to distinguish between the inducing effect of the ammonium 12 present in the medium and volatile ammonia from itself or nearby cultures. To 13 circumvent this problem, and to obtain nitrogen surplus conditions we increased 14 the glutamate concentration of the recipient culture five fold to 170mM. In these 15 conditions, the biofilm inducing effect of volatile ammonia was still observed 16 (data not shown). This demonstrates that the recipient culture is able to sense and 17 respond to volatile ammonia even in the presence of excess nitrogen. We 18 conclude that volatile ammonia is an information carrying molecule in addition to 19 being able to provide nutritive value. 20

We have shown that B. licheniformis cells are capable of sensing other 21 bacteria growing nearby and of responding to this information. This may represent 22 the earliest evolutionary example of olfaction, since it is the biological detection 23 Ammonia is the simplest readily metabolizable nitrogen source. In many 4 environments, nitrogen is often one of the nutrients limiting for bacterial growth, 5 nitrogen use is strictly regulated (24, 25), and an ability to detect its nearby 6 presence would be advantageous. However, it was not observed until now that 7 bacteria can sense the presence of volatile ammonia and respond to this in a 8 coordinated matter. 9

Identification of the sensing mechanism and the signalling pathways 10 controlling subsequent biofilm formation is the next step in this line of research. 11

There is increasing evidence that Amt proteins identified in E. coli and a wide 12 range of bacteria including Bacillus subtilis act as ammonia sensors (26). It is also 13 interesting to note that proteomic data suggest that ammonia sensing may be 14 coupled to the regulation of a wide variety of environmental responses supporting 15 our observations that ammonia is an environmental signal molecule (26). The 16 ability of volatile molecules to affect bacterial growth and biofilm formation will 17 have major implications for our understanding of microbiology in general and of 18 biofilm biology specifically. A better understanding of the mechanisms that 19 trigger biofilm formation may also lead to new approaches to their prevention. 20 Indeed, the discovery of chemotaxis, quorum sensing and more recently 21 was taken using a Canon Powershot G6 digital camera. Below: Absorbance of 1 these cells was measured at OD 595 every 30 minutes for 39 hours and the averaged 2 growth curve of 1 column/8 wells is shown. The absorbance at T = 39 hours is 3 visualized using a color scale ranging from OD 595 0.85 (green) to OD 595 1.70 4 (red). 5 6 2B: confirmation of ammonia as the inducing signal 7

Bacillus licheniformis DSM13 was grown stationary in MSgg medium in the ten 8 middle columns of a clear 96 well plate for 24 hours, in a Fluostar Optima plate 9 reader (BMG labtech, UK) at 30ºC. The cultures were flanked by one column of 10 wells containing an aqueous solution of ammonia at 25mM (row 1) and 2.5mM 11 (row 12). Absorbance was measured at OD 595 every 30 minutes and the averaged 12 growthcurve of 1 column/8 wells was plotted. The absorbance at T = 24 hours 13 was visualized using a color scale ranging from OD 595 0.70 (green) to OD 595 1. 35 14 (red). 15 On the right (rows 11+12) the same supernatant with the pH adjusted to 5.0 with HCl was used. Lowering the pH to 5.0 results in conversion of all free NH3 to NH4+, which is no longer volatile. As such no biofilm induction is observed. The image was taken using a Canon Powershot G6 digital camera. Below: Absorbance of these cells was measured at OD595 every 30 minutes for 39 hours and the averaged growth curve of 1 column/8 wells is shown. The absorbance at T = 39 hours is visualized using a color scale ranging from OD595 0.85 (green) to OD595 1.70 (red).

2B: confirmation of ammonia as the inducing signal Bacillus licheniformis DSM13 was grown stationary in MSgg medium in the ten middle columns of a clear 96 well plate for 24 hours, in a Fluostar Optima plate reader (BMG labtech, UK) at 30ºC. The cultures were flanked by one column of wells containing an aqueous solution of ammonia at 25mM (row 1) and 2.5mM (row 12). Absorbance was measured at OD595 every 30 minutes and the averaged growthcurve of 1 column/8 wells was plotted. The absorbance at T = 24 hours was visualized using a color scale ranging from OD595 0.70 (green) to OD595 1.35 (red).

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  Bacillus licheniformis DSM13 was grown in MSgg medium in the ten 1 central columns of a clear 96 well plate, stationary, for 24 hours, in a Fluostar 2

  compound over a distance. This observation suggests that the ability 1 to sense volatile signals and act on this information to enhance survival evolved 2 first in bacteria. 3

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  communication by physical contact (27) illustrates that bacteria appear to have 22 most of the five senses which include sight (responsiveness to light), touch 23 gene expression), taste (chemotaxis, Quorum Sensing) and 1 now smell. This new dimension to bacterial information exchange also opens up 2 the possibility of using this biochemical pathway as a model system for 3 understanding olfaction and the evolution of smell in higher organisms. 4

1 2Figure 1 :Figure 2 14 2A:

 1114 Figure 1: Induction of biofilm formation in microtitre plates 3

  Figure 1: 1

Figure 1 :

 1 Figure 2: 1 2 3 4

Wiley-VCH Biotechnology Journal

Acknowledgments

(NE/G011206/1). We would like to thank Mike Hall for advice on ammonia 5 chemistry. 6 7

The authors have declared no conflict of interest 8