Luca Federici

Michele Masulli

Daniele Bonivento

Adele Di

Stefano Gianni

Bartolo Favaloro

Carmine Di Ilio

Nerino Allocati
email: n.allocati@dsb.unich.it

Role of Ser11 in the stabilization of the structure of Ochrobactrum anthropi glutathione transferase

Keywords: glutathione, CDNB, 1-chloro-2,4-dinitrobenzene, GdnHCl, guanidine hydrochloride, GST, glutathione transferase, OaGST, Ochrobactrum Ochrobactrum anthropi, bacterial GST, G-site, unfolding, crystallography, circular dichroism

Glutathione transferases (GSTs) are a multifunctional group of enzymes, widely distributed and involved in cellular detoxification processes. In the xenobiotic degrading bacterium Ochrobactrum anthropi, GST is overexpressed in the presence of toxic concentrations of aromatic compounds such as 4-chlorophenol and atrazine.

We have determined the crystal structure of the GST from O. anthropi (OaGST) in complex with GSH. Like other bacterial GSTs, OaGST belongs to the beta class and shows a similar binding pocket for GSH. However, in contrast with the structure of Proteus mirabilis GST, GSH is not covalently bound to Cys 10 but is present in the thiolate form. In our investigation of the structural basis for GSH stabilization we have identified a conserved network of hydrogen-bond interactions, mediated by the presence of a structural water molecule that links Ser 11 to Glu 198 . Partial disruption of this network, by mutagenesis of Ser 11 to Ala, increases the K m for GSH by 15 fold and decreases the catalytic efficiency by 4 fold, even though Ser 11 is not involved in GSH binding. Thermal and chemical induced unfolding studies point to a global effect of the mutation on the stability of the protein and to a central role of these residues in zippering the terminal helix of the C-terminal domain to the starting helix of the Nterminal domain.

INTRODUCTION

Glutathione transferases (GSTs; EC 2.5.1.18) are a family of multifunctional enzymes involved in cell detoxification from xenobiotic compounds [START_REF] Hayes | The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance[END_REF][START_REF] Hayes | Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress[END_REF][START_REF] Armstrong | Structure, catalytic mechanism, and evolution of the glutathione transferases[END_REF][START_REF] Mannervik | Glutathione transferases: structure and catalytic activity[END_REF][START_REF] Hayes | Glutathione Transferases[END_REF]. The classical reaction catalyzed by GSTs is the conjugation of glutathione (GSH) to hydrophobic electrophilic compounds, which generates products that are more easily expelled from the cell [START_REF] Hayes | The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance[END_REF][START_REF] Hayes | Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress[END_REF][START_REF] Armstrong | Structure, catalytic mechanism, and evolution of the glutathione transferases[END_REF][START_REF] Mannervik | Glutathione transferases: structure and catalytic activity[END_REF]. Together with this activity, GSTs also show peroxidase and isomerase activities and are capable of binding several substrates non-catalytically [START_REF] Hayes | The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance[END_REF][START_REF] Hayes | Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress[END_REF][START_REF] Armstrong | Structure, catalytic mechanism, and evolution of the glutathione transferases[END_REF][START_REF] Mannervik | Glutathione transferases: structure and catalytic activity[END_REF]. GSTs are divided into at least three major families of proteins, namely cytosolic, mitochondrial and microsomal GSTs [START_REF] Hayes | Glutathione Transferases[END_REF]. The cytosolic GSTs have been sub-grouped into several divergent classes on the basis of sequence identity, substrate specificity and type of catalyzed reactions [START_REF] Hayes | Glutathione Transferases[END_REF][START_REF] Sheehan | Structure, function and evolution of glutathione transferases: implications for classification of nonmammalian members of an ancient enzyme superfamily[END_REF]. Despite interclass sequence identity being often less than 20%, all cytosolic GSTs are dimeric proteins showing a very conserved fold consisting of two domains: a N-terminal thioredoxin-like domain and C-terminal all helical domain [START_REF] Armstrong | Structure, catalytic mechanism, and evolution of the glutathione transferases[END_REF][START_REF] Sheehan | Structure, function and evolution of glutathione transferases: implications for classification of nonmammalian members of an ancient enzyme superfamily[END_REF]. Two binding sites are located at the domains' interface, the Gsite that accommodates GSH and the H-site where the hydrophobic electrophiles bind [START_REF] Armstrong | Structure, catalytic mechanism, and evolution of the glutathione transferases[END_REF][START_REF] Sheehan | Structure, function and evolution of glutathione transferases: implications for classification of nonmammalian members of an ancient enzyme superfamily[END_REF].

Several GSTs of bacterial origin have been grouped into their own class (known as beta class) and are characterized by a relatively poor conjugation activity as compared to mammalian GSTs [START_REF] Di Ilio | Purification and characterization of three forms of glutathione transferase from Proteus mirabilis[END_REF]. They are capable of binding several antibiotics including tetracycline and rifamycin, show some differences in their dimeric organisation as compared to mammalian GSTs and perform glutaredoxin-like activity [START_REF] Perito | Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis[END_REF][START_REF] Rossjohn | A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications[END_REF][START_REF] Caccuri | GSTB1-1 from Proteus mirabilis: a snapshot of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme[END_REF]. Beta class GSTs are characterized by the presence of a cysteine in the G-site [START_REF] Rossjohn | A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications[END_REF]. Notably GSH forms a mixed disulphide with Cys 10 in the structure of GST from Proteus mirabilis (PmGST) while it is present in the thiolate form in the structure of GST from Burkholderia xenovorans (BxGST) [START_REF] Caccuri | GSTB1-1 from Proteus mirabilis: a snapshot of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme[END_REF][START_REF] Tocheva | Structures of ternary complexes of BphK, a bacterial glutathione S-transferase that reductively dechlorinates polychlorinated biphenyl metabolites[END_REF]. Cys 10 was demonstrated to play a role in the glutaredoxin-like reaction catalyzed by PmGST [START_REF] Caccuri | GSTB1-1 from Proteus mirabilis: a snapshot of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme[END_REF], while its mutation to alanine did not perturb the classical conjugation activity of PmGST, in a fashion similar to the substitution of the catalytic tyrosine or serine of other GST classes, leaving the structural basis of GSH thiolate stabilization in beta class GSTs unclear [START_REF] Casalone | Site-directed mutagenesis of the Proteus mirabilis glutathione transferase B1-1 G-site[END_REF].

Ochrobactrum anthropi is a xenobiotics-degrading bacterium that is able to proliferate in the presence of highly toxic compounds such as phenol, 4-chlorophenol and atrazine and to use them as a carbon source [START_REF] Laura | Effects of atrazine on Ochrobactrum anthropi membrane fatty acids[END_REF][START_REF] Favaloro | Modulation of the glutathione S-transferase in Ochrobactrum anthropi: function of xenobiotic substrates and other forms of stress[END_REF]. The overexpression of the gene encoding a functional GST (OaGST), mainly localized in the periplasm [START_REF] Tamburro | Expression of glutathione S-transferase and peptide methionine sulphoxide reductase in Ochrobactrum anthropi is correlated to the production of reactive oxygen species caused by aromatic substrates[END_REF], is achieved by the treatment with such aromatic compounds pointing to the hypothesis that this enzyme serves in cellular detoxification processes [START_REF] Favaloro | Modulation of the glutathione S-transferase in Ochrobactrum anthropi: function of xenobiotic substrates and other forms of stress[END_REF][START_REF] Tamburro | Bacterial peptide methionine sulphoxide reductase: co-induction with glutathione S-transferase during chemical stress conditions[END_REF].

In this work we have determined the crystal structure of OaGST in complex with GSH and analyzed similarities and differences with other bacterial GSTs. We identify a network of hydrogen-bond interactions, conserved in all beta class GSTs, that zippers the terminal helix of the C-terminal domain to the starting helix of the thioredoxin-like domain. In order to characterize the role of this network in the G-site and overall protein stabilization we have partially disrupted it by mutating Ser 11 to Ala. Our results demonstrate that Ser 11 plays a crucial role in both the stability and functionality of OaGST.

MATERIAL AND METHODS

Expression and purification of wild-type and mutant OaGST enzymes

The recombinant OaGST and S11A mutant enzymes were expressed in Escherichia coli XL1-Blue strains as previously described [START_REF] Favaloro | Molecular cloning, expression and site-directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi[END_REF]. Briefly, E. coli cells were grown overnight at 37°C in Luria-Bertani (LB) medium [START_REF] Sambrook | Molecular cloning: A Laboratory Manual[END_REF], diluted 1:10 and grown in fresh LB medium until the A 550 reached 0.4. To induce gene transcription, IPTG (isopropylβ-D-thiogalactopyranoside) (Sigma-Aldrich, Milano, Italy) was added to a final concentration of 1 mM and the incubation time was prolonged for a further 16 h after switching the temperature to 25°C.

The bacterial cells were collected by centrifugation, washed twice and resuspended in 0.025 M imidazole-HCl, pH 7.4 (buffer A) containing 2 mM dithiothreitol and disrupted by sonication in the cold. The particulate material was removed by centrifugation and the resulting supernatant was subjected to chromatofocusing on a column (i.d. 1.0 cm, height 50 cm) containing polybuffer exchanger PBE 94 (Amersham) equilibrated with buffer A. The column was eluted with polybuffer 74 (Amersham) diluted 1:8, pH 4.0 (flow rate 35 ml/h, fraction volume 2 ml). The peak of activity thus separated was concentrated and dialysed against 10 mM Tris/HCl, pH 7.5 (buffer B), by ultrafiltration in an Amicon apparatus. Concentrated enzyme was further purified by anion-exchange chromatography with a DEAE column (i.d. 1.5 cm, height 11 cm; Bio-Rad Laboratories, Milano, Italy) equilibrated with buffer B.

The enzyme was eluted with a 100 ml linear gradient of 0-0.6 M KCl in buffer B (flow rate 0.5 ml/min, fraction volume 1 ml). The peak containing GST activity was pooled, concentrated, dialysed against 10 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA by ultrafiltration and subjected to further analyses. SDS/PAGE in discontinuous slab gel was performed by the method of Laemmli [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF].

Protein concentration was determined by the method of Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] with γ-globulin as standard.

Crystallization and data collection

Wild-type OaGST was extensively dialyzed against water and concentrated up to 5 mg/ml. Prior to crystallization trials, GSH 10 mM was added to the protein sample.

Crystals were obtained by the hanging drop vapour diffusion method using the following protocol: 1 µl of protein sample was mixed in a cover slip with 1 µl of a reservoir solution containing 2.0 M ammonium sulphate, Tris pH 7.0 100 mM and 200 mM Li 2 SO 4 . The cover slip was sealed on a well containing 1.0 ml of the same reservoir solution and equilibrated at 295 K. Crystals appeared after three days and reached a final size of 0.1x0.1x0.2 mm. The addition of GSH was found to be indispensable for crystal growth. The same or other crystallization conditions failed to produce crystals of the S11A enzyme.

Best data for wild-type OaGST were collected at the ID14-4 beamline of ESRF synchrotron source (Grenoble) equipped with an Oxford cryostream system set at 100 K. Before freezing, crystals were cryoprotected by pouring them in a solution identical to the mother liquor plus 25% glycerol. Complete data were collected to the resolution of 2.1 Å. Data were processed and scaled using DENZO and SCALEPACK, respectively [START_REF] Otwinowski | Processing of X-ray diffraction data collected in oscillation mode[END_REF]. Crystals belong to the space group P6 1 22. Unit cell dimensions together with statistics about the data processing are summarized in Table 1.

Structure solution and refinement

The structure of OaGST was determined by Molecular Replacement using as a search model the structure of E. coli GST monomer (pdb code 1A0F), including all side chains but with the exclusion of the glutathione sulphoxide ligand. The program MOLREP from the CCP4 suite was used [START_REF] Vagin | An approach to multi-copy search in molecular replacement[END_REF]. The optimal solution yielded a single monomer in the asymmetric unit, in accordance to Matthews coefficient calculations, and the physiological dimer was correctly generated by crystallographic symmetry.

With this asymmetric unit composition, the solvent content of the crystal is 49%. This solution was initially refined using rigid body minimization and simulated annealing as implemented in the program CNS [START_REF] Brunger | Crystallography & NMR system: A new software suite for macromolecular structure determination[END_REF]. At this stage electron density maps were visually inspected and clear electron density for GSH was observed. Model building and refinement were carried out iteratively using COOT [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF], to visualize the maps and adjust the model, and restrained refinement as implemented in REFMAC5 [START_REF] Murshudov | Refinement of Macromolecular Structures by the Maximum-Likelihood Method[END_REF].

An area of positive electron density in the difference Fo-Fc map, contoured at 3.0 σ, was found at the dimer interface in exact correspondence with the symmetry axis.

Given its shape and the crystallization conditions, this area was interpreted by adding a sulphate ion to the model. The final model was refined to a R-factor of 18.7% and a Rfree of 23.2 % and contains all 201 residues of the protein, one sulphate ion and 96 water molecules. The final quality of the model is excellent as judged using PROCHECK [START_REF] Laskowsky | PROCHECK-A program to check the stereochemical quality of protein structures[END_REF]. Only Gln 65 has unfavourable stereochemistry according to Ramachandran calculations: this residue is implicated in GSH binding and shows the same stereochemistry in all GSTs determined so far. Notably, Tyr 86 was found to assume two different conformational states in the monomers forming the physiological dimer. Since the dimer is obtained through crystallographic symmetry, Tyr 86 was modelled in two configurations with halved occupancies. Statistics relative to the refinement and the quality of the model are shown in Table 1.

Structural superpositions were performed using the program COOT. Contact maps where calculated using the CONTACT MAP ANALYSIS algorithm as implemented in the SPACE server (http://ligin.weizmann.ac.il/cma). Figures 1 and3 were prepared using PyMOL [START_REF] Delano | The PyMOL Molecular Graphics System[END_REF].

The coordinates and structure factors were deposited within the Protein Data Bank and the accession code is 2NTO.

Enzyme activity

GST activity with CDNB (1-chloro-2,4-dinitrobenzene) was assayed at 25°C according to the method of Habig and Jakoby [START_REF] Habig | Assay for differentiation of glutathione Stransferases[END_REF]. For the enzyme kinetic determinations either CDNB or GSH were held constant at 1 and 5 mM respectively, whilst the concentration of the other substrate was varied (0.1-5 mM for GSH and 0.1-1.6 mM for CDNB). Each initial velocity was measured at least in triplicate. The KaleidaGraph Software package (Synergy Software, Reading, PA, U.S.A.) was used to estimate the Michaelis constant (K m ) and V max values by non-linear regression analysis using the Michaelis-Menten equation.

The dependence of k cat /K m on pH was determined by using the following buffers (0.1 M) at the indicated pH values: Bis-Tris/HCl, from 5.0 to 7.0; and Tris/HCl, from 7.2 to 9.0. The reactions were carried out using saturating GSH (5 mM) and variable CDNB concentrations. The pK a values were obtained by fitting the data to the

equation: log(k cat /K m ) = log[C/(1+[H + ]/K a )
] where C is the upper limit of k cat /K m at high pH [START_REF] Liu | Contribution of tyrosine 6 to the catalytic mechanism of isoenzyme 3-3 of glutathione S-transferase[END_REF].

Measurements of enzyme activity (0.7 µM) as a function of temperature were carried out by incubating the samples at each temperature for 15 min. GST activity was determined at the end of the incubation.

Unfolding studies

To study the dependence of enzyme activity on GdnHCl concentration, wild-type and mutant GST (7 µM) were first incubated for 30 min at 25 °C in 0.1 M potassium phosphate buffer (pH 6.5) containing 1 mM EDTA with 0-4 M GdnHCl. At the end of incubation, each sample was assayed for remaining GST activity in 1 ml of final volume but including the same concentration of GdnHCl as used in the incubation. Equilibrium thermal denaturations were followed on a JASCO circular dichroism (CD) spectrophotometer using a 0.1 cm quartz cuvette (Hellma). Protein concentration was 5 µM. Data for wild type and S11A enzymes were fitted using the equation: In the analysis, the S11A enzyme data were cut at the temperature of 328 K because at this temperature the protein grossly precipitates.

GdnHCl-induced equilibrium denaturations were performed at 298 K recording CD spectra in the interval 215-230 nm. GdnHCl concentration was progressively increased by 0.2 M, from 0 to 4.6 M, and the spectra recorded using a 1.0 cm quartz cuvette (Hellma) and a protein concentration of 1 µM. Assuming a standard two-state model, the GdnHCl-induced denaturation transition for the wild-type enzyme was fitted to the equation:

ΔG d = ΔG 0 -m D-N D
where ΔG 0 is the free energy of folding in water and ΔG d at a concentration D of denaturant, m D-N is the slope of the transition (proportional to the increase in solventaccessible surface area on going from the native to the denatured state). An equation that takes into account the pre-and post-transition baselines was used to fit the observed unfolding transition [START_REF] Santoro | A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range[END_REF].

RESULTS AND DISCUSSION

Overall structure and comparison with beta class GSTs

The structure of OaGST was determined by molecular replacement using the structure of Escherichia coli GST (EcGST) as a search model [START_REF] Nishida | Three-dimensional structure of Escherichia coli glutathione S-transferase complexed with glutathione sulfonate: catalytic roles of Cys10 and His106[END_REF]. One monomer of OaGST is found in the asymmetric unit and the physiological dimer is obtained through crystallographic symmetry (Figure 1A). Statistics about data collection and refinement are listed in Table 1.

OaGST displays the typical overall fold of this super-family of enzymes and is composed by two domains: an N-terminal thioredoxin-like domain (residues 1-76) and a C-terminal α-helical domain (residues 89-201), connected by a linker region.

Like other bacterial GSTs, OaGST may be classified as belonging to the beta class. A structure-based comparison with the other three GSTs of known structure belonging to this class, PmGST, EcGST and the recently solved BxGST [START_REF] Tocheva | Structures of ternary complexes of BphK, a bacterial glutathione S-transferase that reductively dechlorinates polychlorinated biphenyl metabolites[END_REF], reveals relatively high sequence identities, i.e. 34%, 39% and 46% respectively. This is reflected in the rms deviations of equivalent Cαs that are 1.488 Å for OaGST-PmGST, 1.38 Å for OaGST-EcGST and 1.33 Å for OaGST-BxGST. These deviations are smaller than those obtained by comparing OaGST with GSTs representative of other classes. In this case rms deviations range from 1.85 Å for the superposition with the delta class GST from Anopheles gambiae to 2.67 Å for the superposition with the murine pi class GST. Nevertheless these numbers highlight that the overall GST fold is remarkably well conserved from bacteria to mammals, despite very low inter-class sequence identities.

A peculiarity of beta class GSTs is that their dimer interface is mainly polar and close-packed and differs from the more open and hydrophobic V-shaped interface observed in other classes. The relative orientation of the monomers in the beta-class GST dimers is very well conserved as observed by superposing the OaGST dimer with the PmGST, EcGST and BxGST dimers (Figure 1B). Rms deviations for these superpositions are 1.614 Å, 1.418 Å and 1.54 Å, respectively. These values are very close to those obtained by superposing the monomers alone. Interestingly, such a remarkable conservation in the dimer architecture is achieved through a set of interactions that are only partially conserved. This is shown in Figure 2 where interchain contact maps for beta class GSTs are represented. For instance, a number of contacts between residues belonging to the C-terminal domain of both monomers in PmGST and EcGST (lower right in the contact maps) are not conserved in OaGST and partially in BxGST. Conversely in OaGST, a number of contacts between the Nterminal domain of one monomer and the C-terminal domain of the other monomer are not conserved in the other structures (lower left or upper right in the contact maps). Many other differences are also visible throughout the contact maps that provide a fingerprint for each dimer interface. This analysis suggests that, after divergence among the different bacteria, evolution was oriented toward the conservation of the relative orientation of the monomers in the dimers rather than to preserve the chemical nature of the interface.

The G-site

In the structure of OaGST, GSH is stabilized through a number of ionic and H-bond interactions with several residues including Lys 35 , Val 52 through its peptide carbonyl and amide groups, Asn 66 and Gln 65 . Similarly to other beta class GSTs, GSH bound to Biochemical Journal Immediate Publication. Published on 16 Jan 2007 as manuscript BJ20061707 a monomer is hydrogen-bonded to a residue (Asp 103 ) of the other monomer and contributes to the stabilization of the dimer interface in the ligand-bound form. An interesting difference with PmGST is that in the structure of OaGST we do not observe a mixed-disulphide bond between GSH and Cys 10 (Figure 3). This is also true for the structure of BxGST cocrystallized with GSH [START_REF] Tocheva | Structures of ternary complexes of BphK, a bacterial glutathione S-transferase that reductively dechlorinates polychlorinated biphenyl metabolites[END_REF], while the structure of EcGST was obtained in the presence of the GSH analogue, glutathione sulphonate [START_REF] Nishida | Three-dimensional structure of Escherichia coli glutathione S-transferase complexed with glutathione sulfonate: catalytic roles of Cys10 and His106[END_REF]. In OaGST, the distance between the GSH and enzyme Cys 10 cysteinyl groups is 3.11 Å suggesting, together with the shape of the electron density, that the GSH is present in the thiolate form and shares a proton with Cys 10 . The contribution of G-site residues to the thiolate stabilization has been investigated in PmGST and partly in EcGST. In the PmGST structure, three residues were found to be at hydrogen bond distance from the GSH sulphur, namely Cys 10 , Ser 9 and His 106 (in PmGST numbering). Mutation of Cys 10 to Ala in both PmGST and EcGST did not affect the conjugating activities of these enzymes but caused an increase in the GSH pK a [START_REF] Caccuri | GSTB1-1 from Proteus mirabilis: a snapshot of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme[END_REF][START_REF] Inoue | Effect of Cys10 mutation to Ala in glutathione transferase from Escherichia coli[END_REF]. Mutation of Ser 9 to Ala in PmGST caused a moderate loss in specific acitvity but no variation in the pK a [START_REF] Casalone | Site-directed mutagenesis of the Proteus mirabilis glutathione transferase B1-1 G-site[END_REF]. Finally, mutation of His 106 to Ala in PmGST caused a dramatic effect to the catalytic activity but only a moderate shift to the GSH pK a (from 6.4 to 6.7) [START_REF] Allocati | Evaluation of the role of two conserved active-site residues in beta class glutathione S-transferases[END_REF]. By observing the G-site conformation, we note that in OaGST, as well as in BxGST, the topological position corresponding to the PmGST Ser9 is occupied by an alanine that cannot contribute to the thiolate stabilization. Moreover, in OaGST, His 105 occupies a different position from its topologically equivalent His 106 of PmGST, BxGST and EcGST. This is due to the absence of charge repulsion with the following Lys 107 that is topologically replaced by an alanine in OaGST. As a consequence, OaGST His 105 Nε1 and Nε2 side chain nitrogens are at a distance of 4.58 Å and 4.26 Å, respectively, from the GSH sulphur, as compared to an average distance of ~3.3 Å in the other GSTs, and none of them has a favourable geometry to interact with the GSH sulphur. Thus in the OaGST structure two possible contributors to GSH thiolate stabilization are either absent or in an unfavourable position.

Nevertheless, the thiolate stabilization achieved in OaGST is more effective than in PmGST since the pK a value for GSH in OaGST is 5.96 (Table 2) while in PmGST is 6.4 [START_REF] Caccuri | GSTB1-1 from Proteus mirabilis: a snapshot of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme[END_REF]. Thus it seems plausible to hypothesize that, in OaGST, Cys 10 plays an important role in both the stabilization of the GSH thiolate and in defining the conjugation activity.

Cys 10 was also found to be crucial for the glutaredoxin-like activity of PmGST [START_REF] Caccuri | GSTB1-1 from Proteus mirabilis: a snapshot of an enzyme in the evolutionary pathway from a redox enzyme to a conjugating enzyme[END_REF]. This enzyme is always recovered in the oxidized state after purification, with Cys 10 covalently linked to GSH in a mixed disulphide [START_REF] Rossjohn | A mixed disulfide bond in bacterial glutathione transferase: functional and evolutionary implications[END_REF]. Interestingly this is not the case for both OaGST and BxGST that are purified and crystallized in the reduced state. As we have noted before the main difference at the G-site between PmGST and OaGST/BxGST is the presence in the former of a serine at position 9, establishing a H-bond interaction with the GSH sulphur, that is replaced by an alanine in OaGST, BxGST and EcGST. A possible role of PmGST Ser 9 in defining the increased tendency of this enzyme towards the formation of a mixed disulphide as compared to the other beta class GSTs may thus be hypothesized but remains to be established.

Mutation of Ser 11 affects the enzyme activity and G-site stability

By analyzing a sequence alignment of all bacterial GSTs available in the database (data not shown) we observed that, together with Cys 10 , Ser 11 is also completely conserved. Mutation of Ser 11 to Ala resulted in a marked loss in activity [START_REF] Favaloro | Molecular cloning, expression and site-directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi[END_REF]. The position of this residue in the structure of OaGST attracted our attention since Ser 11 is the first residue of helix 1 and its side chain is not part of the G-site but it points to the opposite direction with respect to Cys 10 side chain (Figure 3). The distance between the Ser 11 hydroxyl group and the GSH sulphur is 6.43 Å, which excludes a direct role in GSH binding and stabilization. A closer analysis of the structure highlights the presence of a network of H-bond interactions that are conserved in all beta-class GSTs (Figure 3). Three residues take part in this network: Ser 11 is linked to Glu 198 through a water molecule that is hydrogen-bonded to both residues. Glu 198 is in turn also hydrogen-bonded to His 15 . The structural role played by the water molecule is witnessed by its thermal B-factor (23.65 Å 2 ) that is in the range of main chain atoms B-factors. The effect of the Ser 11 to Ala mutation on enzyme function was tested and the specific activities and kinetic constants with CDNB as a second substrate for wildtype and S11A OaGST are shown in Table 2. The effect of the mutation is exerted mainly at the G-site. The K m for GSH is considerably increased (by 15-fold) while the k cat /K m is decreased by 5-fold and the specific activity decreased by 3-fold.

Interestingly the k cat is increased by 4-fold. Taken together these data suggest that the mutation considerably lowers the affinity for GSH and the overall efficiency of the enzyme but favours the release of the product once the reaction has taken place. Since Ser 11 does not bind the GSH, this might, in a first instance, be ascribed to an increased mobility and/or destabilization of the G-site in the mutated enzyme.

The effect on the CDNB kinetic constants is exerted mainly on the K m but is less evident in agreement with the observation that this mutation is far from the H-site (Table 2). Interestingly a long-range effect is observed also in the GSH thiolate stabilization with a slight decrease of the pK a from 5.96 to 5.84.

To investigate a possible effect on the structural stability of the enzyme G-site we also followed the conjugation of CDNB by GSH as a function of temperature and GdnHCl concentration. The thermal stability of the S11A enzyme is compromised: while the wild-type enzyme retains more than 80% of its functionality at 50 °C, the S11A enzyme is already almost inactive at 45 °C (Figure 4A). Accordingly, also the effect of GdnHCl is markedly different for the S11A enzyme as compared to the wild-type (Figure 4B). At [GdnHCl]=0.5 M the S11A enzyme has completely lost its activity while the wild-type enzyme still retains 50% of its functionality.

Global effect of the H-bond network on OaGST stability

Next we wanted to establish if the effect of the mutation on enzyme kinetics might be only ascribed to a local effect on the G-site conformation or rather to a global effect on the enzyme stability. Unfortunately every attempt to crystallize this mutant failed, however CD spectra of the wild-type enzyme and the mutated enzyme in the 205-250 nm interval are completely superimposable (inset in Figure 5) suggesting that the structure of the mutant is native-like in physiological conditions.

Figure 5 shows thermal denaturation of the wild-type and mutated enzymes. While the wild-type and S11A enzymes display similar native baselines, confirming that the mutated enzyme is fully folded in physiological temperature range, the transition to the unfolded species is shifted at lower temperature for the mutated enzyme. This is quantitatively confirmed by fitting the experimental data with a two state model. Our analysis suggests that the T m for the mutated enzyme is decreased by 7 °C (from 56.68 °C to 49.75 °C) and that the overall destabilization caused by the mutation may be estimated as ΔΔG ~1.8 kcal/mol. We also followed the denaturation of the enzyme in the presence of increasing amounts of GndHCl. In Figure 6 the wild-type enzyme is still fully folded. A closer investigation of the observed denaturation profiles reveals that, while a simple two-state transition can be assigned to the wild-type data, a more complex denaturation is monitored for the S11A mutant. Such behaviour may be consistent with the accumulation of an unfolding intermediate. Taken together, both chemical and thermal unfolding data clearly suggest that a remarkable destabilization is associated with the S11A mutation, i.e. above 60% of the total free energy of the enzyme, the overall stability of the wildtype protein being about 3 kcal/mol.

In this work we have structurally and functionally characterized the GST from O. anthropi, a xenobiotics degrading bacterium capable of growing in the presence of highly toxic compounds such as 4-chlorophenol and atrazine. On the basis of its structural similarity with previously characterized GSTs, OaGST may be classified as belonging to the beta class.

Analyzing the structure of OaGST, we have identified a previously unrecognized network of hydrogen bonds, located at the boundaries of the G-site, but distinct from it, that links the first helix of the N-terminal thioredoxin-like domain to the terminal helix of the C-terminal domain. We have shown that the single mutation S11A dramatically alters the catalytic capabilities of the enzyme towards the model substrate CDNB. This is likely the result of the crucial role played by this network in stabilizing the overall fold of the protein, as inferred from chemical and temperature induced unfolding studies. Since this H-bond network is conserved in beta class GSTs but not in GSTs belonging to other classes we conclude that, within the framework of the very conserved GST fold, evolution found different solutions to achieve structural stabilization in the various GST classes.

=

  where y N and y D represent the CD signals of the native and denatured state, respectively; T m is the melting temperature; ΔH Tm is the change in enthalpy upon denaturation at the melting temperature and the Δc p is the heat capacity change upon unfolding.

  the denaturation profiles for the wild-type and the mutated enzyme are shown and a different behaviour is observed. Again a clear transition for the mutated enzyme is visible at very low GndHCl concentration where Biochemical Journal Immediate Publication. Published on 16 Jan 2007 as manuscript BJ20061707 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1 .

 1 Figure 1. A) Ribbon representation of the physiological dimer of OaGST. Bound GSH molecules are shown in sticks. A sulphate ion, found at the interface between monomers, exactly marks the position of the crystallographic 2-fold axis. B) Structural superposition of GST dimers belonging to the beta class: OaGST (light blue), PmGST (magenta), EcGST (orange) and BxGST (green).

Figure 2 .

 2 Figure 2. Contact maps for beta class GSTs. Interactions between residues belonging to different monomers in the dimers are shown as squares. Several differences are observed throughout the maps, even though the orientations of the monomers in the various GSTs dimers are conserved.

Figure 3 .

 3 Figure 3. Close up view of OaGST in the GSH surroundings. Cys 10 is not covalently linked to the GSH. Residue Ser 11 is oriented in the opposite direction and interacts with Glu 198 via a structural water molecule. Glu 198 is also hydrogen-bonded to His 15 .These residues are conserved in beta class GSTs.

Figure 4 .

 4 Figure 4. A) OaGST residual activity with CDNB as a function of temperature and B) OaGST residual activity with CDNB as a function of increasing concentrations of GdnHCl. Wild-type enzyme (). S11A enzyme ().

Figure 5 .

 5 Figure 5. Thermal melt of OaGST followed by circular dichroism. Data were fitted with a two-state model. Straight line represents wild-type OaGST data; dashed line represents S11A OaGST data. The overall destabilization caused by this single point mutation is estimated to be 1.8 kcal mol -1 . Inset shows CD spectra for wild-type (straight line) and S11A (dashed line) in the interval 200-250 nm.

Figure 6 .

 6 Figure 6. GdnHCl-induced equilibrium denaturation of OaGST followed by circular dichroism. Wild type data are represented with filled circles, S11A data are shown with empty circles. Wild-type data were fitted with a two-state model. This model could not be applied satisfactorily to the S11A data, due to the appearance of an unfolding intermediate. Overall stability of the protein is estimated to be 2.86 kcal/mol.

Figure 5 Biochemical

 5 Figure 5

Table 2 .

 2 Specific activity and kinetic constants for OaGST and the S11A mutant with CDNB as second substrate. The values are presented as means + S.D. for at least three independent determinations.

	_______________________________________________________________________________________________________________________
					GSH			CDNB
				______________________________	____________________________
	Specific activity	K m	k cat	k cat /K m	K m	k cat	k cat /K m	pK a	CDNB
	Enzyme (µmol min	-1 mg	-1 )	(mM)	(s -1 )	(mM -1 s -1 )	(mM)	(s

Biochemical Journal Immediate Publication. Published on 16 Jan 2007 as manuscript BJ20061707 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 16 Jan 2007 as manuscript BJ20061707 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

We thank the beamline scientists at the ESRF (Grenoble, France) for setting up the experimental beamline for x-ray diffraction. This work was partially supported by grants from Ministero dell'Universita' e della Ricerca.


Georgia A Kotzia

Katerina Lappa

Nikolaos E Labrou
email: lambrou@aua.gr.

Tailoring structure-function properties of Lasparaginase: Engineering resistance to trypsin cleavage. by

Keywords: L-asparaginase, hydrolase, enzyme engineering, leukaemia, PEG, PEGylation, proteolytic resistance

Bacterial L-asparaginases (L-ASNases) catalyze the conversion of L-asparagine to Laspartate and ammonia, and are widely used for the treatment of acute lymphoblastic leukemia (ALL). In the present study we describe an efficient approach, based on protein chemistry and protein engineering studies, for the construction of trypsinresistant PEGylated L-ASNase from Erwinia carotovora (EcaL-ASNase). Limited proteolysis of EcaL-ASNase with trypsin was found to be associated with a first cleavage of the peptide bond between Lys53 and Gly54, and then a second cleavage at the Arg206-Ser207 position of the C-terminal fragment, peptide 54-327, showing that the initial recognition sites for trypsin are Lys53 and Arg206. Site-directed mutagenesis of Arg206 to His followed by covalent coupling of methoxypolyethylene glycol succinate N-hydroxysuccinimide ester (mPEG-SNHS) to the mutant enzyme resulted in an improved modified form of EcaL-ASNase that retains 82% of the original catalytic activity, exhibits enhanced resistance to trypsin degradation, and has higher thermal stability compared to the wild-type enzyme.

INTRODUCTION

L-Asparaginase (L-ASNase) catalyzes the hydrolysis of the non-essential amino acid L-Asn to L-Asp and ammonia. L-ASNase is widely used for the treatment of haematopoetic diseases such as acute lymphoblastic leukemia (ALL). The antineoplastic activity results from depletion of the circulating pools of L-Asn by Lasparaginase [START_REF] Lee | L-asparaginase from Erwinia carotovora. An improved recovery and purification process using affinity chromatography[END_REF][START_REF] Keating | L-asparaginase and PEG asparaginase-past, present, and future[END_REF]. Unlike normal cells, malignant cells can only synthesize L-Asn slowly and are dependent on an exogenous supply [START_REF] Lee | L-asparaginase from Erwinia carotovora. An improved recovery and purification process using affinity chromatography[END_REF][START_REF] Keating | L-asparaginase and PEG asparaginase-past, present, and future[END_REF]. In contrast, normal cells are protected from Asn starvation by their ability to produce this amino acid [START_REF] Duval | Comparison of Escherichia coli-asparaginase with Erwinia-asparaginase in the treatment of childhood lymphoid malignancies: results of a randomized European organization for research and treatment of cancer-children's Leukemia group phase 3 trial[END_REF]. Thus, unlike conventional cancer therapy, L-ASNase therapy is highly selective.

The main restrictions to the use of L-ASNase as a therapeutic agent include its premature inactivation and rapid clearance, thus necessitating frequent injections to maintain therapeutic levels, and several types of side effects ranging from mild allergies and development of immune responses to anaphylactic shock, which may be life-threatening [START_REF] Mashburn | Tumor inhibitory effect of Lasparaginase from Escherichia coli[END_REF][START_REF] Marlborough | Comparative study on conformational stability and subunit interactions of two bacterial asparaginases[END_REF][START_REF] Moola | Erwinia chrysanthemi L-asparaginase: epitope mapping and production of antigenically modified enzymes[END_REF][START_REF] Soares | Effects of polyethylene glycol attachment on physicochemical and biological stability of E. coli L-asparaginase[END_REF]. To date, the most promising approach to extend the half-life and reduce the immunogenicity and antigenicity of therapeutic proteins is to covalently couple them to poly(ethylene glycol) (PEG) [START_REF] Burnham | Polymers for delivering peptides and proteins[END_REF][START_REF] Veronese | Peptide and protein PEGylation: a review of problems and solutions[END_REF][START_REF] Veronese | PEGylation, successful approach to drug delivery[END_REF]. In this process (PEGylation), PEG is attached to the therapeutic protein by covalent linkage to residues located at the surface of the native protein [START_REF] Matsushima | Modification of E. coli asparaginase with 2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-S-triazine (activated PEG2)[END_REF]. PEGylation of proteins usually results in masking of some surface sites, increasing the molecular size and enhancing steric hindrance. Thus, attachment of PEG to proteins reduces their immunogenicity, improves pharmacokinetics [START_REF] Veronese | Bioconjugation in pharmaceutical chemistry[END_REF][START_REF] Harris | Effect of PEGylation on pharmaceuticals[END_REF], plasma half-life, and protects the proteins against proteolytic cleavage [START_REF] Inada | Biomedical and biotechnological applications of PEG-and PM-modified proteins[END_REF][START_REF] Matsushima | Bioconjugates of proteins and polyethylene glycol: potent tools in biotechnological processes[END_REF][16][START_REF] Baran | Solid-phase enzyme modification via affinity chromatography[END_REF]. The in vivo activity of the modified protein can sometimes be enhanced due to longer half-life in the circulation after conjugation, even though there may be some reduction in in vitro activity.

PEGylation has been extensively used to modify various proteins such as adenosine deaminase [START_REF] Hershfield | PEG-ADA: an alternative to haploidentical bone marrow transplantation and an adjunct to gene therapy for adenosine deaminase deficiency[END_REF], superoxide dismutase [START_REF] Beauchamp | A new procedure for the synthesis of polyethylene glycol-protein adducts; effects on function, receptor recognition, and clearance of superoxide dismutase, lactoferrin, and alpha 2-macroglobulin[END_REF], asparaginase [START_REF] Kodera | Stabilization of L-asparaginase modified with comb-shaped poly (ethylene glycol) derivatives, in vivo and in vitro[END_REF], urokinase [21], hirudin [START_REF] Humphries | High and constant plasma levels of tissue plasminogen activator and PEG-hirudin can be achieved by subcutaneous delivery[END_REF], hemoglobin [START_REF] Ajisaka | Modification of human hemoglobin with polyethylene glycol: a new candidate for blood substitute[END_REF], interferon [START_REF] Wedemeyer | Polyethylene glycol-interferon: Current status in hepatitis C virus therapy[END_REF], etc. In the USA, there are currently six polymer-modified proteins that have already been approved by the FDA and about a dozen more in various phases of clinical development [START_REF] Pavlou | Recombinant protein therapeuticssuccess rates, market trends and values to 2010[END_REF].

The toxicity of L-ASNase is also partially attributable to the glutaminase activity of these enzymes [START_REF] Howard | Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase[END_REF]. Thus, L-ASNases with high specificity for L-Asn and low-to-negligible activity against L-Gln are reported to be less troublesome during the course of anti-cancer therapy [27]. The interest in L-asparaginase from Erwinia Biochemical Journal Immediate Publication. Published on 21 Feb 2007 as manuscript BJ20061708 © 2007 Biochemical Society carotovora arose from the fact that it shows reduced glutaminase activity, and it is therefore believed to have fewer side effects when used in anti-cancer therapy [28].

All asparaginases are homotetramers with 222-symmetry and a molecular mass in the range of 140-150 kDa, with a highly conserved overall fold [29][30][31]. They are composed of four identical subunits denoted A, B, C, and D [START_REF] Kozak | Crystallization and preliminary crystallographic studies of five crystal forms of Escherichia coli Lasparaginase II (Asp90Glu mutant)[END_REF]. Each monomer consists of about 330 amino acid residues that form 14 β-strands and 8 α-helices, which are arranged in two easily identifiable domains: the larger N-terminal domain and the smaller C-terminal domain. These are connected by a linker consisting of ~20 residues. Each of the four active sites is located between the N-and C-terminal domains of two adjacent monomers. Thus, the asparaginase tetramer can be treated as a dimer of dimers, as the active site is derived from subunits A and C or from subunits B and D.

For many pharmaceutical proteins, enzymatic proteolysis makes maintaining appropriate serum levels problematic. The use of L-ASNase in therapy is limited by its premature inactivation. For example, it has been reported that serum trypsin levels increase significantly 10 and 20 days after the start of ASNase therapy [START_REF] Shimizu | Increased serum trypsin and elastase-1 levels in patients undergoing Lasparaginase therapy[END_REF][START_REF] Guo | Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis[END_REF]. Here we report an efficient approach for the construction of water soluble and trypsinresistant PEGylated L-ASNase from Erwinia carotovora, based on protein chemistry and protein engineering.

MATERIALS AND METHODS

MATERIALS

L-Asn and L-Gln were obtained from Serva (Heidelberg, Germany). αketoglutaric acid, Sepharose CL6B, trypsin and methoxypolyethylene glycol succinate N-hydroxysuccinimide ester (mPEG-SNHS, mol wt ~5000) from Sigma Co. (St Louis, USA). NADH (disodium salt, grade II, ca. 98 %) and crystalline bovine serum albumin (fraction V), were purchased from Boehringer Mannheim (Germany).

Nessler's reagent and glutamate dehydrogenase (GDH) were obtained from Fluka (Taufkirchen, Germany) and Sephadex G-25 from Pharmacia (Sweden). All primers were synthesized and purified by MWG-biotech AG (Ebersberg, Germany). TOPO cloning kit and all other molecular biology reagents were from Invitrogen (USA).

METHODS

Cloning, expression, and purification of EcaL-ASNase

L-asparaginase from Erwinia carotovora NCYC 1526 (EcaL-ASNase) was cloned into a T7 expression vector (pCR ® T7/CT-TOPO ® ), as described by Kotzia and Labrou [START_REF] Kotzia | Cloning, expression and characterisation of Erwinia carotovora L-asparaginase[END_REF]. The resulting expression construct pT7ASNase was used to transform competent BL21(DE3)pLysS E. coli cells, in which the synthesis of EcaL-ASNase was induced by 1 mM IPTG. The produced enzyme was purified from the other proteins using affinity chromatography on L-Asn-Sepharose CL6B column, according to Kotzia and Labrou [START_REF] Kotzia | Cloning, expression and characterisation of Erwinia carotovora L-asparaginase[END_REF].

Kinetic analysis of mutants and PEGylated enzymes

Enzyme assays were performed at 37 °C at a Hitachi U-2000 double beam uv-vis spectrophotometer carrying a thermostated cell holder (10 mm pathlength). Activities were measured by determining the rate of ammonia formation, by coupling with glutamate dehydrogenase (GDH), according to Balcao et al. [START_REF] Balcao | Structural and functional stabilization of L-asparaginase via multisubunit immobilization onto highly activated supports[END_REF]. The final assay volume of 1 ml contained 71 mM Tris-HCl buffer, pH 8.0; 1 mM Asn; 0.15 mM αketoglutaric acid; 0.15 mM NADH; 4 units GDH, and sample containing EcaL-ASNase activity. Alternatively, the rate of ammonia formation was measured at 37 ºC using the Nessler's reagent. One unit of Asparaginase activity is defined as the amount of enzyme that liberates 1 µmol of ammonia from Asn per minute at 37 °C.

Protein concentration was determined at 25 °C by the method of Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] using bovine serum albumin (fraction V) as standard.

Steady-state kinetic measurements were performed in 0.1 M Tris-HCl buffer, pH 8.0 (or 8.2 for L-Gln), by varying the concentration of the substrate (L-Asn and L-Gln).

The kinetic parameters were calculated by non-linear regression analysis of experimental steady-state data. Kinetic data were analyzed using the computer program GraFit (Erithacus Software Ltd., [START_REF] Leatherbarrow | GraFit, Version[END_REF]). Kinetic experiments were repeated three times to verify the results.

Limited proteolysis of EcaL-ASNase by trypsin

EcaL-ASNase, in 10 mM Tris-HCl pH 7.4, was subjected to trypsin digestion.

Proteolysis was initiated by adding trypsin (dissolved in 1 mM HCl, containing 1 mM CaCl 2 ) to EcaL-ASNase at a L-ASNase/trypsin ratio of 1:35 (w/w). Samples were withdrawn at selected incubation intervals and the trypsinolysis was stopped by freezing the samples with liquid nitrogen. All samples were lyophilized and then analyzed by SDS-PAGE.

Site-directed mutagenesis, expression and purification of mutants

Site-directed mutagenesis of Arg206 was carried out by overlap extension using the polymerase chain reaction (PCR) [START_REF] Ho | Sitedirected mutagenesis by overlap extension using the polymerase chain reaction[END_REF]. The pairs of oligonucleotide primers used in the PCR reactions for the Arg206His or Arg206Gln mutation were as follows: the first pair 5'-ATG TTT AAC GCA TTA TTC GTT GTT GTT TTT G -3' (P 1 ) and 5'-AAA CAC GGA NTG CGT GGT GTG AAC-3' (P 2 ), and the second pair 5'-GTT CAC ACC ACG CAN TCC GTG TTT-3' (P 3 ) and 5'-TTA AGC TTT TAA TAA GCG TGG AAG TAA TCC -3' (P 4 ). Sites of mutation are indicated in italics. The expression construct pT7ASNase [START_REF] Kotzia | Cloning, expression and characterisation of Erwinia carotovora L-asparaginase[END_REF] encoding the wild-type EcaL-ASNase was used as template DNA in all mutagenesis reactions. After completion of the PCR reactions (using primers P1, P2 and P3, P4), the PCR products were digested with DpnI to eliminate parental DNA, and purified by agarose-gel electrophoresis.

Following gel extraction, the fragments containing either mutation (Arg206His or Arg206Gln) were used in another PCR reaction as templates using P 1 and P 4 primers to amplify the entire mutated gene. The latter was TOPO ligated into a T7 expression vector (pCR ® T7/CT-TOPO ® ) and recombinant plasmids were isolated. Site-directed mutagenesis of Lys30 was carried out as described above. The pairs of oligonucleotide primers used in the PCR reactions for the Lys30Ala mutation were as follows:

5'-ACCACTGGGTATGCAGCGGGTGCGC-3' and 5'-CGCACCCGCTGCATACCCAGTGGT-3'. All mutations were verified by DNA sequencing.

Mutated expression constructs were used to transform competent BL21(DE3)pLysS E. coli cells. The E. coli cells were grown at 37 o C in 1 l LB medium containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol. The synthesis of the mutated enzymes was induced by the addition of 1 mM IPTG when the absorbance at 600 nm was 0.6-0.8. Five hours after induction, cells were harvested by centrifugation at 8000 r.p.m. and 4 ºC for 20 min, resuspended in 5 mM potassium phosphate buffer, pH 7.5, sonicated and left at 4 ºC under agitation. After 1h, cells were centrifuged at 10000g for 5 min and the supernatants were collected. Cell-free extracts of E. coli transformants showed high asparaginase activity. The collected supernatants were applied to L-Asn-Sepharose CL6B affinity column (2 ml, 1.5 x 1.5 cm I.D.)

previously equilibrated with 5 mM potassium phosphate buffer, pH 7.5 [START_REF] Kotzia | Cloning, expression and characterisation of Erwinia carotovora L-asparaginase[END_REF]. Nonadsorbed protein was washed off with 20 ml equilibration buffer. Bound L-ASNase (Arg206His or Arg206Gln) was eluted with equilibration buffer containing 10 mM L-Asn (6 ml). Collected fractions (1 ml) were assayed for L-ASNase activity and protein (A 280 or Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]). The results from a typical purification run showed for the Arg206Gln, 75.4-fold purification with 95.3% yield, for the Arg206His mutant showed 119.7-fold purification with 96.5% yield and for the Lys30Ala mutant 110fold purification with 97% yield.

Modification of the

Succinylation

EcaASNase was succinylated with succinic anhydride as described in [START_REF] Shifrin | L-Asparaginase from Escherichia coli B. Succinylation and subunit interactions[END_REF] with the following modifications: succinic anhydride crystals were added to 10 mM final concentration over a period of 1 h to a solution of 0.2 mg of the enzyme in 0.1 M potassium phosphate buffer (pH 8.5) at 4°C. The solution was constantly stirred and maintained at pH 8.5 by the addition of 0.01 M NaOH. Modification reaction was allowed to proceed for 6h at 4 ºC, before it was stopped by the addition of 12 mM glycine, pH 9. The extent of reaction with lysyl residues was determined from titration of free amino groups with trinitrobenzene sulfonic acid [START_REF] Snyder | An improved 2,4,6trinitrobenzenesulfonic acid method for the determination of amines[END_REF].

Limited proteolysis with trypsin of Arg206His and PEGylated enzymes

Agr206His, PEG-EcaL-ASNase and PEG-Arg206His, previously equilibrated in 10 mM Tris-HCl pH 7.4, were subjected to trypsin digestion. Proteolysis was carried out using L-ASNase/trypsin ratio of 1:35 (w/w). All samples were lyophilized and then analyzed by SDS-PAGE. Gels run with a Laemmli buffer system [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. The intensities of bands were quantified with the 1D Image Analysis Software (Version 3.5, Kodak) analysis software. The intensity change versus time was fit to the first-order rate equation 1:

I = I 0 e -t kobs equation 1
where I is the intensity at time t, I 0 is the intensity at t=0, and k obs is a first-order rate constant.

Thermal stability of EcaL-ASNase, Arg206His and PEGylated enzymes

Irreversible thermal inactivation of EcaL-ASNase, Arg206His and PEGylated enzymes was monitored by activity measurements. Samples of the enzymes, in 50 mM potassium phosphate buffer pH 8.5, were incubated at different temperatures (35 ºC-50 ºC) for 7.5 min. Subsequently the samples were assayed for residual activity, using the coupled enzyme assay method described above. The T m values were determined from the plots of relative inactivation (%) versus temperature (°C). T m value is the temperature at which 50% of the initial enzyme activity is lost after heat treatment.

The time course of thermal inactivation of EcaL-ASNase, Arg206His and PEGylated enzymes, in 50 mM potassium phosphate buffer pH 8.5, was studied at 37°C. The rate of inactivation was followed by periodically removing samples for assay of enzymatic

MALDI-TOF MS

The samples were desalted with ZipTip µ-C18 (Millipore, USA). A saturated solution of sinapinic acid in water/acetonitrile (50/50, v/v, 0.1 % trifluoroacetic acid) was used as the matrix solution. One µL of the sample and matrix mixture was spotted into a well of the sample plate and dried on the sample holder. Mass spectra were taken with a Voyager System 6322 (Applied Biosystems). Mass calibration was performed using bovine serum albumin, apomyoglobin and cytochrome c.

N-terminal amino acid sequence analysis

N-terminal amino acid sequence analysis of the proteolytic products resulting from the trypsin digestion of the EcaL-ASNase was carried out on a gas-phase Applied 542

Biosystems Protein sequencer, model 470A, equipped with an on-line phenylthiohydantoin analyzer.

Electrophoresis

SDS polyacrylamide gel electrophoresis was performed according to the method of Laemmli [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] on a slab gel containing 12.5% (w/v) polyacrylamide (running gel) and 2.5% (w/v) stacking gel. The protein bands were stained with Coomassie Brilliant Blue R-250 or with silver nitrate.

RESULTS and DISCUSSION

Limited proteolysis of EcaL-ASNase

To obtain information about the protease-sensitive cleavage sites in EcaL-ASNase, limited proteolysis with trypsin was carried out. To follow the time course of formation and disappearance of proteolytic fragments, samples were withdrawn from the tryptic digestion mixture at intervals and subjected to SDS-PAGE for separation of the proteolytic products (Figure 1A). In particular, Lys53 is located at the short β-sheet formed by residues Asn51-Val57.

Arg206 is located at a flexible segment, formed by residues Asp201-Asp221, which connects the N-terminal and C-terminal domains (Figure 1B).

Our results thus showed that in order to engineer resistance to trypsin cleavage into EcaL-ASNase, two main amino acid targets should be considered: Lys53 and Arg206. Taking into account that Lys53 is located at a surface accessible to the solvent and that this residue can be efficiently protected through chemical modification with succinimide esters of poly(ethylene glycol), Arg206 was selected for site-directed mutagenesis.

Site-directed mutagenesis of EcaL-ASNase

On the basis of limited proteolysis and computational analysis studies, Arg206

was selected for site-directed mutagenesis. This residue was mutated to the conserved residues His and Gln, which are not recognized targets of trypsin. Kinetic analysis of the mutants was carried out to evaluate the effect of each mutation on substrate binding and catalysis, and the results are given in Table 1. Arg206His showed differences in K m and specific activity values for L-Asn when compared to the wildtype enzyme. In particular, mutant Arg206His showed an increase in K m value of about 6.4-fold for L-Asn and a slight decrease in specific activity (~9%) compared to the wild-type enzyme. Arg206Gln gave low expression levels and worse kinetic properties compared to Arg206His (data not shown); thus, it was not considered further. The mutant Arg206His was subjected to limited proteolysis with trypsin, under the same conditions as used for the wild-type enzyme, in order to verify the absence of the ~25 kDa fragment resulting from trypsin digestion. SDS-PAGE analysis of the tryptic digestion mixture showed that after 90 min of incubation, no band corresponding to ~25 kDa was formed (Figure 2).

PEGylation of EcaL-ASNase and the Arg206His mutant enzyme

PEGylation has become one of the most extensively used techniques, and has attracted increasing attention for enhancement of the therapeutic potential of proteinbased pharmaceuticals [START_REF] Hershfield | PEG-ADA: an alternative to haploidentical bone marrow transplantation and an adjunct to gene therapy for adenosine deaminase deficiency[END_REF][START_REF] Beauchamp | A new procedure for the synthesis of polyethylene glycol-protein adducts; effects on function, receptor recognition, and clearance of superoxide dismutase, lactoferrin, and alpha 2-macroglobulin[END_REF][START_REF] Kodera | Stabilization of L-asparaginase modified with comb-shaped poly (ethylene glycol) derivatives, in vivo and in vitro[END_REF][21][START_REF] Humphries | High and constant plasma levels of tissue plasminogen activator and PEG-hirudin can be achieved by subcutaneous delivery[END_REF][START_REF] Ajisaka | Modification of human hemoglobin with polyethylene glycol: a new candidate for blood substitute[END_REF][START_REF] Wedemeyer | Polyethylene glycol-interferon: Current status in hepatitis C virus therapy[END_REF][START_REF] Pavlou | Recombinant protein therapeuticssuccess rates, market trends and values to 2010[END_REF]. Recent advances in recombinant technology and the development of functional group-selective reactive PEG derivatives have led to the creation of PEGylated proteins or mutants with markedly enhanced in vitro and in vivo characteristics and with minimal loss of bioactivity compared to their unmodified equivalents [START_REF] Harris | Effect of PEGylation on pharmaceuticals[END_REF].

Various factors including inherent nucleophilicity, solvation, protein conformation, and also local electronic and pK a effects determine the actual reactivity of the potential PEGylation sites [START_REF] Roberts | Chemistry for peptide and protein PEGylation[END_REF]. The reaction chemistry and the length of PEG chains are the minimum set of variables that can determine the biological activity and stability of the modified protein [START_REF] Soares | Effects of polyethylene glycol attachment on physicochemical and biological stability of E. coli L-asparaginase[END_REF][START_REF] Harris | Effect of PEGylation on pharmaceuticals[END_REF]. In the present work, the Nhydroxysuccinamide derivative of PEG (mPEG-SNHS) was employed. mPEG-SNHS allows linkage between PEG and exposed Lys residues and/or the N-terminal amino group in proteins under mild conditions of pH and temperature [START_REF] Matsuyama | Phospholipase D modified with a polyethylene glycol derivative[END_REF]. This approach is suitable for EcaL-ASNase because the enzyme contains twenty lysine residues in addition to the N-terminal amino acid. None of these lysine residues are located in regions of the protein that are directly involved in substrate binding and catalysis [START_REF] Ho | Sitedirected mutagenesis by overlap extension using the polymerase chain reaction[END_REF]. The effect of PEGylation (at 4.28 mg mPEG-SNHS/25 µg protein) on the enzyme activity of both the wild-type enzyme and the Arg206His mutant was evaluated by measuring the enzyme activity after the completion of the modification reaction. The PEG-EcaL-ASNase retained 78% of its initial activity and the PEG-Arg206His retained 82%. The tendency to lose activity can be attributed to the steric hindrance caused by the conjugation of PEG to the free amino groups on the protein surface, which prevents L-Asn from approaching the active site of the enzyme [START_REF] Zhang | Chemical modification of Lasparaginase from Escherichia coli with a modified polyethyleneglycol under substrate protection conditions[END_REF].

The modification of EcaL-ASNase and Arg206His with mPEG-SNHS led to a 3.6-to 5-fold increase in K m value for L-Asn and L-Gln, respectively. The increased K m value for L-Asn, although undesirable, is still within the range of acceptability for therapeutic applications [START_REF] Asselin | Measurement of serum L-asparagine in the presence of L-asparaginase requires the presence of an L-asparaginase inhibitor[END_REF]. On the other hand, the increase in K m value by 5-fold (24.8 mM) for L-Gln is desirable, since the ability of therapeutic preparations of Lasparaginases to hydrolyze L-Gln has been implicated to cause several side effects

[27].

To analyze whether the effect of PEGylation on K m value (K m =0.09 ±0.03 mM for L-Asn) is the result of modification of a specific Lys residue or from the structure/chemistry of PEG we employed protein chemistry and site-directed mutagenesis studies. Complete chemical modification of EcaL-ASNase by succinic anhydrite results to a succinylated enzyme (fifteen residues succinylated per subunit) which retains unaltered its K m , in agreement with the results reported by Shifrin et al., [START_REF] Shifrin | L-Asparaginase from Escherichia coli B. Succinylation and subunit interactions[END_REF]. It is apparent that extensive succinylation does not destroy enzyme function and therefore this finding indicates that none of the Lys residue is essential for catalytic activity or substrate binding.

In silico analysis of residues' accessibility in EcaL-ASNase showed that the seven, out of twenty Lys residues, that exhibit high accessible surface area are: Lys30, Lys45, Lys53, Lys145, Lys216, Lys257, Lys269. The other Lys residues are either more buried or lie at the subunit interface. These seven residues are probable the targets of chemical modification by mPEG-SNHS. Analysis of the crystal structure of the enzyme showed that none of these lysine residues are located in regions of the protein implicated in substrate binding and catalysis with the only exception that of Lys30 which lies at the flexible loop (residues 10-40, Figure 1B) near to the enzyme active site [29,31,[START_REF] Kotzia | Cloning, expression and characterisation of Erwinia carotovora L-asparaginase[END_REF]. Site-directed mutagenesis of Lys30 to Ala gave a mutant enzyme with kinetic parameters very close to the wild-type enzyme (K m = 0.075 ± 0.03 mM for L-Asn). Therefore the results from site-directed mutagenesis and from extensive chemical modification by succinic anhydrite point to the conclusion that the influence of PEGylation on K m value is an indirect effect and may be described as originating from the overall shielding effect of the PEG chains rather than from the modification of a specific Lys residue. This conclusion is also supported by the observation that Arg206His mutant enzyme, after PEGylation, exhibits K m values or L-Asn and L-Gln comparable to that of the wild-type enzyme.

Trypsin digestion of PEGylated enzymes

To evaluate the effect of modification with PEG on the rate of trypsinolysis of PEG-EcaL-ASNase and the PEG-Arg206His mutant enzyme, limited proteolysis experiments were carried out under the same conditions as those reported for the unmodified enzymes. SDS-PAGE analysis of the products of the trypsinolysis reaction showed that after 90 min of reaction, trypsin had completely digested PEG-EcaL-ASNase, whereas PEG-Arg206His showed considerable resistance to proteolysis (Figure 3). The rate of breakdown of both enzymes was monitored by quantifying the amount of intact enzyme on SDS-PAGE gels as a function of incubation time. The decrease in intensity of the enzyme band was fitted to a firstorder rate equation. The observed rates of digestion (k obs ) were found to be 1.54 ± 0.18 and 0.1 ± 0.12 for PEG-EcaL-ASNase and PEG-Arg206His, respectively. The protective effect of PEGylation has been described as originating from a shell of PEG chains surrounding the protein, with the shielding effect of the PEG chains contributing to the inaccessibility of the enzyme to trypsin [START_REF] Veronese | Bioconjugation in pharmaceutical chemistry[END_REF][START_REF] Harris | Effect of PEGylation on pharmaceuticals[END_REF].

Thermal stability of EcaL-ASNase, Arg206His and PEGylated enzymes

To determine the structural stability of EcaL-ASNase, Arg206His, and the PEGylated enzymes, heat inactivation studies were carried out. The enzymes were incubated at different temperatures and subsequently assayed for residual activity (Figure 4). The corresponding T m values are listed in Table 2.

The time course of thermal inactivation of EcaL-ASNase, Arg206His, and the PEGylated enzymes was also investigated at the temperature of therapeutic importance, i.e. 37ºC. The results indicated that the PEGylated enzymes are particularly stable at 37ºC whereas the EcaL-ASNase and Arg206His mutant enzyme have significantly lower thermostability. Kinetic analysis of the thermal inactivation at 37ºC gave linear plots (Figure 5), from which the first-order inactivation rate constants k in could be calculated according to equation 3:

log(%remaining activity) = 2.303 · k in · t equation 3
The results are summarized in Table 2. The PEGylated enzymes showed a dramatic improvement in thermal stability at 37ºC, which is about 30.7-fold and 55.6-fold higher (for PEG-EcaL-ASNase and PEG-Arg206His, respectively) than for non-PEGylated enzymes. It is believed that PEGylation usually enhances the thermal stability of proteins by sterically blocking degradation pathways induced by hydrophobic interactions [START_REF] Bailon | Poly(ethylene glycol)-conjugated pharmaceutical proteins[END_REF], and by causing non-specific steric hindrance of the intermolecular interactions that are involved in thermal instabilities [START_REF] Hinds | Effects of PEG conjugation on insulin properties[END_REF].

Conclusion

A short half-life in serum is a common trait of most proteins being used or developed for therapeutic purposes. Proteolysis is being increasingly acknowledged to play a significant role in the clearance of therapeutic proteins. Thus, the design of novel

  EcaL-ASNase and mutant Arg206His with mPEG-SNHS and purification of the PEGylated enzymesPurified L-ASNase (EcaL-ASNase and/or its mutant Arg206His, 25µg), in 50 mM potassium phosphate buffer pH 8.5, was mixed with different amounts (0.28mg, 0.7mg, 1.15mg, 2.14mg and 4.28mg) of mPEG-SNHS (methoxypolyethylene glycol succinate N-hydroxysuccinimide ester, MW 5000 Da) in order to determine the necessary amount for complete modification. Modification reactions were allowed to proceed for 22h at 4 ºC, before they were stopped by the addition of 12 mM glycine, pH 8. The effect of PEGylation on enzyme activity was evaluated by taking samples at various time intervals during the modification reaction and measuring the residual activity, using the coupled enzyme assay method described above. The purification of PEG-EcaL-ASNase and/or PEG-Arg206His was carried out using size exclusion chromatography on a Sephadex G-25 column (9.1 ml, 1.5 x 5 cm I.D.), equilibrated with 10 mM Tris-HCl buffer, pH 7.4 and eluted with the same buffer at a flow rate of 0.5 ml/min. The samples were analyzed by SDS-PAGE and MALDI-TOF MS as described below. Residues' accessibility was determined in silico using the WHAT IF software package[40].

  Figure 1B. Both cleavage sites are positioned at the external surface of EcaL-ASNase.

  Purified L-ASNases (wild-type and the Arg206His mutant) were subjected to PEGylation using different amounts (0.28-4.28 mg) of mPEG-SNHS in order to determine the optimum [enzyme]/[mPEG-SNHS] ratio for complete enzyme modification. The reactions were allowed to proceed for a long period of time(22 h, 4°C) in order to make sure that complete PEGylation would be occurred. SDS-PAGE analysis of the products of the modification reactions showed that the amount of mPEG-SNHS necessary for complete modification of the enzymes was 4.28 mg mPEG-SNHS/25 µg protein. This ratio promotes the formation of a single modified form of the enzyme with subunit molecular mass of approximately 70.5 kDa, as Biochemical Journal Immediate Publication. Published on 21 Feb 2007 as manuscript BJ20061708 © 2007 Biochemical Society determined by SDS-PAGE analysis (Figure 3, see 'control' lanes). The other concentrations used gave a mixture of different forms with molecular masses between 36.5 to 70.5 kDa, due to incomplete modification (data not shown). The lowest amount used, 0.28 mg mPEG-SNHS, had no significant effect on the enzyme's molecular weight. MALDI-TOF was used to determine precisely the number of PEG molecules attached to the wild-type and mutant enzymes. Analysis of the mass spectra gave mass/charge ratios of 69,492.3 and 69,472.4 for the modified wild-type and mutant enzymes (subunit MW), respectively. Given the molecular weight of PEG and L-ASNase, the molecular weights of the modified enzymes corresponded to hepta-PEG-modified L-ASNases.

  proteins that are resistant to digestion, represents important strategy for the creation of next-generation therapeutic products with improved pharmacological properties. In the present study, the EcaL-ASNase was genetically modified by site-directed mutagenesis and chemically modified with mPEG-SNHS. The modified form of the mutant enzyme (PEG-Arg206His) was found to have improved proteolytic stability, good catalytic activity for L-Asn, and high structural stability. This enzyme may be tested further on animals and/or humans in order to create a new drug for future therapeutic use.ACKNOWLEDGEMENTThis work was financially supported by the Hellenic General Secretariat for Research and Technology: Operational Program for Competitiveness, Joint Research and Technology Program.

Figure 1 A

 1 Figure 1 A. Time course of tryptic digestion of EcaL-ASNase Analysis was carried out by SDS-PAGE. Proteolysis was initiated by adding trypsin to the enzyme at an L-ASNase/trypsin weight ratio of 1:35 (w/w). Conditions are shown above lanes. A sample taken immediately after the addition of trypsin to EcaL-ASNase was used as control. The experiment was performed three times to verify the results. B. Structural representations of limited proteolysis sites and critical PEGylation site Lys30, Lys53 and Arg206 are shown in space-filling mode and are labelled. The figure was created based on the crystal structure of E. carotovora L-ASNase (PDB code 1ZCF) [29].

Figure 2

 2 Figure 2 Time course of tryptic digestion of Arg206His mutant enzyme Analysis was carried out by SDS-PAGE. Proteolysis was initiated by adding trypsin to the mutant enzyme at Arg206His/trypsin weight ratio of 1:35 (w/w). Conditions are shown above lanes. A sample taken immediately after the addition of trypsin to Arg206His mutant enzyme was used as control. The experiment was performed three times to verify the results.

Figure 3

 3 Figure 3 Time course of tryptic digestion of PEGylated enzymesAnalysis was carried out by SDS-PAGE. Conditions are shown above lanes. A sample taken immediately after the addition of trypsin to purified PEGylated enzymes were used as control. The experiment was performed three times to verify the results.

Figure 4

 4 Figure 4 Thermal inactivation curves of PEGylated and non PEGylated enzymesThe residual activities of the enzymes were measured after heat treatment at various temperatures for 7.5 min. EcaL-ASNase, (•); Arg206His mutant enzyme, (□); PEG-EcaL-ASNase, (■); and PEG-Arg206His mutant enzyme, (○). The data represent a mean of three independent experiments.

Figure 5

 5 Figure 5 Time course of inactivation of PEGylated and non PEGylated enzymes at 37 ºC Time course of inactivation at 37 ºC of EcaL-ASNase, (□); Arg206His mutant enzyme (▲); PEG-EcaL-ASNase (■); and PEG-Arg206His mutant enzyme (•). The rate of inactivation was followed by periodically removing samples for assay of enzymatic activity. The data represent a mean of three independent experiments.

Figure 3 97Figure 5 Biochemical

 35 Figure 2

Biochemical Journal Immediate Publication. Published on 21 Feb 2007 as manuscript BJ20061708 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 21 Feb 2007 as manuscript BJ20061708 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 21 Feb 2007 as manuscript BJ20061708 © 2007 Biochemical Society activity

  . Observed rates of inactivation (k in ) were deduced from plots of log (% of remaining activity) versus time (h). Thermal inactivation experiments were repeated three times to verify the results.

Biochemical Journal Immediate Publication. Published on 21 Feb 2007 as manuscript BJ20061708 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 21 Feb 2007 as manuscript BJ20061708 © 2007 Biochemical Society

Table 1 Kinetic parameters of PEGylated and non-PEGylated enzymes

 1 Steady-state kinetic measurements were performed at 37 o C. All initial velocities were determined in triplicate. The kinetic parameter K m was calculated by non-linear

	regression analysis of experimental steady-state data using the GraFit (Erithacus
	Software Ltd.) program [38]. The specific activity was estimated as % of the wild-
	type EcaL-ASNase [35].
	Substrates	Enzymes	Specific Activity	K m (mM)
			(% of EcaL-
			ASNase)
		EcaL-ASNase	100	0.085 ± 0.02*
		Arg206His	90.9 ± 1.9	0.55 ± 0.09
	L-Asn	PEG-EcaL-ASNase	78.0 ± 5.0	0.31 ± 0.05
		PEG-R206His	74.5 ± 2.7	0.36 ± 0.07
		EcaL-ASNase	100	6.80 ± 1.32*
		Arg206His	137.5 ± 3.5	4.94 ± 0.58
	L-Gln	PEG-EcaL-ASNase	92.0 ± 2.6	24.69 ± 5.55
		PEG-R206His	83.6 ± 1.7	24.84 ± 4.90
	* According to Kotzia and Labrou [35]

Table 2 T m (ºC) and k in (h -1 , at 37 ºC) values of PEGylated and non-PEGylated enzymes.

 2

	Enzymes	T m (ºC)	k in (h -1 )
			x 10 -3
	EcaL-ASNase	38.9±0.45	347.2±44.6
	Arg206His	38.8±0.84	361.6±45.7
	PEG-EcaL-ASNase	42.9±0.25	11.3±0.8
	PEG-Arg206His	43.1±0.45	6.5±0.9

Biochemical Journal Immediate Publication. Published on 21 Feb 2007 as manuscript BJ20061708 © 2007 Biochemical Society

© 2007 Biochemical Society