Ronan M Kelly

Hans Leemhuis

Henriëtte J Rozeboom

Niels Van Oosterwijk

Bauke W Dijkstra

Lubbert Dijkhuizen
email: l.dijkhuizen@rug.nl

Elimination of competing hydrolysis and coupling side reactions of a cyclodextrin glucanotransferase by directed evolution

Keywords: CGTase, cyclodextrin glucanotransferase, epPCR, error-prone polymerase chain reaction, GH13, glycoside hydrolase family 13, MBS, maltose binding site, Tabium, Thermoanaerobacterium thermosulfurigenes strain protein evolution, amylase, CGTase, protein stability, side reactions, saccharide Biochemical

EM1; pNPG7, 4-nitrophenyl--Dmaltoheptaoside-4-6-O-ethylidene

Introduction

Enzymes are essentially a product of evolution. Continuous evolution of enzymes results in intermediates partially retaining the initial ancestral function while catalysing new function [1]. Over several evolutionary phases, aided by selective pressure, new enzyme function dominates while ancestral function decreases to a mere side reaction [2][3][START_REF] Afriat | The latent promiscuity of newly identified microbial lactonases is linked to a recently diverged phosphotriesterase[END_REF]. This evolutionary phenomenon is clearly evident among members of the glycoside hydrolase family 13 (GH13), retaining a multitude of minor side reaction specificities and activities [START_REF] Stam | Dividing the large glycoside hydrolase family 13 into subfamilies: towards improved functional annotations of alpha-amylase related proteins[END_REF]. Members of GH13 act on starch, glycogen and related oligo-and polysaccharides, representing the largest family of glycoside hydrolases. Their active site regions share high sequence similarity embedded in a TIM ( / ) 8 barrel structural fold [START_REF] Janecek | -amylase family: molecular biology and evolution[END_REF], indicating evolutionary diversification from a common enzyme ancestor. All family members either hydrolyze or transglycosylate -glucosidic linkages to produce -anomeric mono-and oligo-saccharides; catalysis proceeds via a double displacement mechanism with initial formation of a covalent glycosyl intermediate [START_REF] Uitdehaag | X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the alpha-amylase family[END_REF]. It is therefore the type of acceptor substrate utilised in the second half of the reaction which determines enzyme reaction specificity, a water molecule in the case of -amylases and a hydroxyl group of a sugar substrate for the glucanotransferases.

Despite highly specific primary activities of GH13 enzymes, many glucanotransferase enzymes also catalyze hydrolysis as a side reaction. Extensive analysis of cyclodextrin glucanotransferases (CGTase) revealed that this side reaction has a considerable impact on final product yields. CGTases primarily catalyze the glucanotransferase reaction of cyclization, for the formation of circular -

(1,4)-linked oligosaccharides (cyclodextrins) (Fig. 1) [START_REF] Uitdehaag | Enzymatic circularization of a malto-octaose linear chain studied by stochastic reaction path calculations on cyclodextrin glycosyltransferase[END_REF]. These enzymes also exhibit, to a lesser extent, -amylase-like activity, hydrolysing starch into short linear saccharides (Fig. 1). The build-up of short saccharides from starch hydrolysis promotes yet another CGTase side reaction, coupling (Fig. 1). In the coupling reaction cyclodextrin rings are cleaved and transferred to (short) saccharide molecules to yield linear products [START_REF] Van Der Veen | Engineering of cyclodextrin glycosyltransferase reaction and product specificity[END_REF]. While most CGTases generally exhibit low hydrolytic rates, Thermoanaerobacterium thermosulfurigenes strain EM1 (Tabium) CGTase displays unusually high hydrolytic activity, but still low compared to -amylases [START_REF] Kelly | Conversion of a cyclodextrin glucanotransferase into an alpha-amylase: assessment of directed evolution strategies[END_REF].

By determining the structural/molecular factors responsible for the high hydrolytic rates of Tabium CGTase, we aimed at significantly reducing this side activity.

Reduced hydrolytic rates would effectively limit the breakdown of cyclodextrins via the coupling reaction, thus enhancing the enzyme's applicability for industrial applications [START_REF] Qi | Cyclodextrin glucanotransferase: from gene to applications[END_REF].

Experimental Procedures

Bacterial strains, plasmids and protein production -Escherichia coli strain MC1061 was used for DNA manipulations and library screening. CGTase proteins were produced and purified using Bacillus subtilis strain DB104A carrying plasmid pCScgt-tt, as described [START_REF] Leemhuis | Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase: mechanism and kinetics of inhibition by acarbose and cyclodextrins[END_REF]. Plasmid carrying strains were grown on LB medium at 37 C in the presence of kanamycin (50 g/ml for E. coli DNA manipulations -Mutants were constructed in pCScgt-tt as described [START_REF] Leemhuis | Mutations converting cyclodextrin glycosyltransferase from a transglycosylase into a starch hydrolase[END_REF] and verified by DNA sequencing (BaseClear, Leiden, the Netherlands). Single mutants S77P and W239R were constructed using the following oligonucleotides: S77P, Microtitre plates were then incubated at 60 ºC and the amount of -cyclodextrin formed was measured after 2 h by the addition of a 10 µl sample to 100 µl phenolphthalein solution [START_REF] Vikmon | Rapid and simple spectrophotometric method for determination of microamounts of cyclodextrins[END_REF]. Samples were degassed prior to the scan.

HPLC
The enzyme concentration used was 6.9 µM in 10 mM sodium acetate buffer at pH 5.5 [START_REF] Leemhuis | Improved thermostability of Bacillus circulans cyclodextrin glycosyltransferase by the introduction of a salt bridge[END_REF].

Results

Generation of low hydrolytic

Catalytic rates of wild-type and mutant

CGTases -Hydrolytic rates of selected variants S77P, W239R and W239L were lowered compared to wild-type CGTase (Table 2). While the W239R and W239L mutants displayed decreases of 27% and 37% in hydrolytic rates, the S77P variant appeared most effective, lowering the hydrolytic activity 15-fold compared to wild-type (Table 2). Initial -cyclization rates remained similar to wild-type CGTase for the W239 mutants while the S77P variant displayed a 1.8-fold reduction. The overall cyclization/hydrolysis ratio for the S77P mutant increased 8.3-fold compared to wildtype (Table 2). For the disproportionation reaction an approximate 2-fold reduction in k cat was observed for processing the blocked pNPG 7 substrate for the S77P and W239R variants (Table 3). An increase in K M for pNPG 7 was also noted for the mutants despite the absence of direct interactions with the blocked substrate. The S77P and W239R mutations reduced catalytic efficiencies for the breakdown of all three cyclodextrins ( , and ) in the coupling reaction over 8 and 1.5-fold, respectively, compared to wild-type (Table 4).

Product profiles of wild-type and mutant

CGTases -For wild-type CGTase the degradation of -and -cyclodextrins surpassed production after 6 h from starch (Fig. 2). The increased rate of cyclodextrin breakdown coincided with an increase in short linear saccharide production (Fig. 2).

The W239L variant showed a reduced capacity in the production of short linear saccharides while both -, -andcyclodextrin formation remained similar to that of wild-type (Fig. 2). Similarly, for the W239R mutant the amount of total short saccharides did not surpass -, -orcyclodextrin formation at any stage. A minor reduction in -and -cyclodextrin breakdown was noted for this variant compared to wild-type. In contrast, the S77P variant showed a continual increase incyclodextrin production till 9 h with no detectable degradation of -cyclodextrin thereafter (Fig. 2). Despite lower initial cyclodextrin formation, the amount of -,and -cyclodextrins produced at 58 h by the S77P variant, was 32 ( ), 81 ( ) and 57 ( ) % higher than wild-type. Production of short saccharides remained extremely low throughout the 58 h incubation for S77P, 7.5-fold lower than that of wild-type (Fig.

2).

Stability of wild-type and mutant CGTases -

To investigate whether the selected mutations affected enzyme stability, the CGTase variants were denatured by heat using differential scanning calorimetry (DSC). Both the wild-type and mutant proteins displayed irreversible thermal unfolding patterns. The S77P mutant displayed a similar apparent melting temperature as the wild-type with a 1 °C difference (Fig. 3). Both the W239L and W239R variants were, however, severely destabilised, lowering the apparent melting temperature by 15 and 21 °C, respectively, compared to wild-type (Fig. 3).

Structures -Two crystal structures of the S77P mutant were determined at a resolution of 1.6 Å (Table 1), one with and one without a heptasaccharide analog bound at the active site. In the acarbose and maltohexaose soaked crystal electron density was present for seven covalently linked sugar residues, at subsites -4 to +3 in the active site, with the valienamine moiety bound at -1 and the 6deoxyglucose at +1, indicating a complete acarbose positioned from -1 to +3, coupled with a maltotriose at the -2 site (Fig. 4). prior to saccharide binding [START_REF] Leemhuis | The fully conserved Asp residue in conserved sequence region I of the -amylase family is crucial for the catalytic site architecture and activity[END_REF] (Fig. 5).

From the crystal structure of the unliganded S77P mutant it appears that the Glu-258 side chain is orientated almost 1 Å further away from where the glycosidic oxygen of a substrate would be, than in wild-type (Fig. acceptor subsites (Fig. 4). This library has been made previously [START_REF] Kelly | Conversion of a cyclodextrin glucanotransferase into an alpha-amylase: assessment of directed evolution strategies[END_REF] and yielded a mutant CGTase with an 11-fold higher hydrolytic activity than wild-type. Library analysis of 1,500 clones revealed that over 80% of the variants had strongly impairedcyclization activity with no variant retaining over 5% wild-type -cyclization activity while reducing hydrolytic activity.

Mutations at the acceptor sites were therefore not capable of increasing the cyclization versus hydrolysis performance of CGTase. Direct substitution of residues within this area resulted in either a strong increase in hydrolysis or a dramatic loss of overall activity (Fig. 6). Thus residues of the acceptor subsites have optimally evolved to favour the use of saccharide acceptors over water, lowering the ancestral activity of hydrolysis while favouring transglycosylation activity (Fig. 6). This is a further indication that in order to lower enzyme side reactions, mutations of second shell active site residues are required to induce subtle conformational changes within the active site.

Specialization of glucanotransferase function from evolutionary intermediates

within GH13 -Many glucanotransferase enzymes of GH13 display large variations in hydrolysis side reaction rates (Fig. 6). As (B) Superimposition of the wild-type and S77P active sites with a G6 or G7 ligand bound, respectively. Wild-type Tabium CGTase residues are coloured white and S77P mutant residues dark grey. Hydrogen bond interactions are indicated as black dashed lines. Active site subsites shown are numbered -1 to +2. Figure 5 was created using PyMOL [START_REF] Delano | The PyMOL Molecular Graphics System[END_REF].

  Previous protein engineering studies, aimed at improving the cyclodextrin size specificity of CGTases, have been of limited success. A double mutant, Y89D/S146P, at the substrate binding sites of Bacillus circulans 251 CGTase, increased -cyclodextrin production two-fold [12]. Replacement of Ala-233 in Bacillus clarkii 7364 CGTase by basic amino acids enhanced -cyclodextrin forming activity over 4-fold in the neutral pH range [13], while substitution of Tyr-54 in the amylomaltase from Thermus aquaticus reduced hydrolytic activity but also enhanced cyclization activity significantly [14]. To generate CGTase variants with a low hydrolytic activity, we applied directed evolution, introducing random mutations throughout the cgt gene by error-prone (ep) PCR, followed by site-specific saturation mutagenesis. Parallel screening for high cyclization and reduced hydrolytic activity on starch yielded three mutations, with no direct interactions with substrate. The crystal structure of the most effective mutant, S77P, revealed subtle conformational changes of a network of amino acids at the active site responsible for the strongly reduced side reaction rates with retention of primary catalytic function.

and 5 g

 5 /ml for B. subtilis). Purity and molecular weight of CGTase proteins were checked by SDS-PAGE. Enzyme concentrations were determined using the Bradford reagent from Bio-Rad (München, Germany) and bovine serum albumin as standard.

5 '

 5 -T TGG ATA CCT CAG CCT GTA G-'3; W239R, CAT ATG CCG TTT GGA CGG CAG AAG-'3. For site-specific saturation mutagenesis, Ser-77 and Trp-239 of Tabium CGTase were replaced by all nineteen other amino acids residues, using the following oligonucleotides: S77X 5'-T TGG ATA NNS CAG CCT GTA G-'3; W239X, CAT ATG CCG TTT GGA NNS CAG AAG-'3 and pCScgt-tt as PCR template. The combined saturated mutagenesis library, A231X/F260X, was previously constructed in the same manner [10]. Underlined region of oligo`s indicate where the nucleotide substitution were introduced. N is A+G+C+T, S is G+C and X is any amino acid residue. Error-prone PCR mutagenesis -The cgt gene was amplified from pCScgt-tt with the primers For1 (NcoI), 5'-GGC TTT TCA GCC CTG CCC AGG CCA TGG-'3 and Rev1 (KpnI), 5'-GTT TAC AAT TAC GGT ACC TGT ACT AGA-'3. Restriction sites are underlined. PCR mixtures (50 l) contained: 1x Taq DNA polymerase buffer, 1 mM MgSO 4 , 0.005 mM or 0.01 mM MnCl 2 , 0.6 mM of each dNTP, 0.07 M of each primer, 20 ng template and 2.5 units Taq DNA polymerase (Roche) [10]. PCR reactions were performed for 25 cycles: 30 sec 94 C, 40 sec 54 C, and 2 min 72 C. The PCR products were restricted with NcoI and KpnI, and the resulting fragment (2100 bp) was extracted from agarose gel (QIAquick Gel Extraction Kit; Qiagen) and cloned in pCScgt-tt, replacing the wild-type cgt gene. Enzyme assays -All enzyme assays were performed in 10 mM sodium citrate buffer at pH 6.0 and 60º C. -Cyclodextrin forming activity was determined by incubating 1.3 -2.6 nM of enzyme with a 2.5% (w/v) solution of partially hydrolysed potato starch with an average degree of polymerisation of 50 (Paselli SA2; AVEBE, Foxhol, The Netherlands). The amount ofusing 1% (w/v) soluble potato starch (Sigma-Aldrich) and 26 -66 nM of enzyme. The disproportionation 4-nitrophenyl--D-maltoheptaoside-4-6-Oethylidene (pNPG7; Megazyme, Wicklow, Ireland) and 0.5 -20 mM maltose as donor and acceptor substrates, respectively. ) as acceptor substrate. Linear products formed were converted to glucose with amyloglucosidase (Sigma, Zwijndrecht, the Netherlands) and the amount of glucose formed was determined with the GOD-PAP reagent (Roche, Almere, the Netherlands). High-throughput screening of CGTase variants -E. coli MC1061 transformed with the library were plated on LB agar plates, and the resulting colonies were transferred to 200 l of LB medium in 96-well microtiter plates using the Q-pix robot (Genetix, New Milton Hamsphire, U.K.) followed by incubation overnight (750 rpm) at 37º C. For starch hydrolysis, 25 l of each culture was transferred to a second 96 well plate containing 25 l of bacterial protein extraction reagent (Pierce, Rockford, IL) per well to lyse the cells. Subsequently, 200 l of 1% (w/v) soluble starch (Lamers and Pleuger, Wijnegen, Belgium) in 10 mM sodium citrate buffer (pH 6.0) was added, and the microtiter plates were incubated at 60 ºC. After 6 h the amount of reducing sugars formed was measured using an adapted version of the Nelson-Somogyi assay [18]. In parallel, -cyclization activity was measured by addition of 50 µl of cell lysate to 200 µl of 1% (w/v) partially hydrolysed potato starch (Paselli SA2).

  with a Centricon-30K device to 80 -106 µM in 10 mM sodium acetate, pH 5.5. Large crystals were grown from 17 -20 % saturated ammonium sulphate at room temperature, 100 mM HEPES buffer, pH 7.6 (or 100 mM EPPS buffer, pH 7.8, or 100 mM Tris-HCl buffer, pH 8.0), like the wild-type enzyme[START_REF] Wind | Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1[END_REF].Before data collection the crystal was soaked in 25% glycerol and directly flash cooled.Another crystal was soaked with 2% (w/v) acarbose, 4% (w/v) maltohexaose and 25% (v/v) glycerol in 20% saturated ammonium sulphate for 25 min followed by flash cooling. Diffraction data were collected at beamline ID14-3, ESRF, Grenoble, France, at 100 K and at a wavelength of 0.931 Å and processed using MOSFLM and SCALA from the CCP4 suite (Collaborative Computational ProjectNumber 4, 1994).Data collection statistics are shown in Table1. The structure of wild-type Tabium CGTase (PDB code 1A47) with all waters and the maltohexaose inhibitor removed was used as a starting model. Rigid body and restrained refinement were done with REFMAC5 in a standard way [21], followed by TLS refinement[22;23]. Ligands were manually placed in sigmaA-weighted 2Fo-Fc and Fo-Fc electron density maps using the program Coot[START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF], water molecules were automatically picked with Coot. In the native and sugar bound structures 12 amino acids have been built with double conformations. The quality of the models was checked using the program MolProbity[START_REF] Davis | MolProbity: all-atom contacts and structure validation for proteins and nucleic acids[END_REF]. The atomic coordinates and the structure factor amplitudes have been deposited with the RCSB Protein Data Bank with PDB codes 3BMV and 3BMW, respectively.

  CGTase variants -Genetic diversity was introduced in Tabium CGTase by epPCR. Twelve thousand CGTase clones were subsequently screened in a parallel manner for decreased starch hydrolytic activity and highcyclization activity using microtitre based plate assays. Five strongly and three slightly improved single mutants were selected. Sequencing of these mutants revealed a S77P mutation in all five strongly improved variants and a W239R mutation in the three slightly improved variants. Both Ser-77 and Trp-239 residues are strongly conserved amongst bacterial and archaeal CGTases, and are replaced only in two hypothetical CGTases (data not shown). Saturated mutagenesis was subsequently carried out at Ser-77 and Trp-239 in an effort to reduce hydrolytic activity even further and retain or raise -cyclization activity. Six hundred clones were screened for both saturated mutant libraries, S77X and W239X. Selection followed by sequencing of five variants of the S77X library revealed the S77P mutation in each case. Sequencing of six selected variants revealed W239R (4x) and W239L (2x) mutations from the W239X library.

7

  Biochemical Journal Immediate Publication. Published on 21 Apr 2008 as manuscript BJ20080353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080353 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Addition of sugar residues to acarbose is commonly observed in CGTase crystal structures[7;26;27]. Electron density was also found for a maltose molecule near Tyr-633 at maltose binding site 3 in the Edomain. Replacement of Ser-77 by Pro showed no alteration in the backbone conformation of residues 75 to 117, compared to the liganded wild-type (PDB Code 1A47). The main chain conformation was not affected by the S77P mutation as the phi/psi angles of Ser77 are -58/158, and those of Pro77 are -63/166. There were also no significant changes observed in temperature factors of the wild-type compared to the average temperature factors of the variants (15.8/20.5 Å 2 for wild-type enzyme and 7.9/7.3 Å 2 for the S77P mutant; the structures of the two variants were determined at different temperatures). However, the Tyr-101 side chain has a double conformation in the unliganded S77P variant. The conformation of the Arg-228 side chain is also altered in the S77P variant, in both structures with and without the ligand, when compared to the native structure. Discussion Effects of the S77P mutation on CGTase side reactions -By applying directed evolution to Tabium CGTase, we identified two mutants with single mutations (S77P and W239R) remote from the active site with strongly reduced hydrolysis side reaction rates on starch. Structural analysis of the mutant CGTase S77P helped to elucidate the effect of these distant mutations on the evolution of side reaction specificity of glucanotransferase enzymes. The S77P residue is positioned at the beginning of a long loop, connecting residues Trp-75 of strand 2 and Thr-117 of helix 2 of the ( / ) 8 barrel structure of the catalytic domain (Fig. 4). Ser-77 is located over 10 Å away from the catalytic nucleophile Asp-230 and 8.7 Å from the nearest saccharide bound at the donor subsites (Fig. 5). While there is no direct interaction between S77P and the substrate, the 75-117 loop contains two essential amino acids, Tyr-101 and Trp-102, that stabilize the substrate bound at donor subsites -1/-2 via hydrophobic stacking and hydrogen bonding interactions, respectively [20]. In the wild-type structure, Ser-77 forms a water mediated hydrogen bonding interaction with Tyr-101. This interaction is lost in the S77P mutant and results in a lack of stabilisation of the Tyr-101 side chain. Alteration of the side chain conformation of Tyr-101, as seen from its double conformation in the unliganded S77P variant, directly affects the hydrogen bonding interaction with the neighbouring Arg-228 residue. The Arg-228 side chain attains a new conformation to retain the hydrogen bonding interaction with the distorted Tyr-101 side chain. This in turn affects the orientation of the acid/base catalyst Glu-258 side chain, which has a hydrogen bond interaction with Arg-228,

5 )

 5 . Slight distortion of the Glu-258 side chain by Arg-228, may therefore require saccharide binding at the acceptor subsites to induce a more catalytically competent conformation of its side chain. This induced side chain repositioning would allow for effective protonation of the leaving saccharide during substrate cleavage and selective deprotonation of the acceptor molecule prior to -1,4 bond formation [29]. The use of water as an acceptor molecule, however, would likely lack the necessary interactions with the active site residues to force the favourable positioning of the Glu-258 side chain for efficient deprotonation. In agreement with this induced-fit mechanism, recent structural studies of the amylomaltase of Thermus thermophilus revealed that the acid/base catalyst Glu-340 only assumes its catalytic competent conformation upon binding of an efficacious saccharide acceptor [of 5000:1 (Fig. 6) [31]. The Trp-239 residue is essential for both CGTase activity and stabilisation -The second mutant identified from our screening, residue W239R, is located at the start of the fourth -helix of the ( / ) 8 barrel (Fig. 4), following the loop (residues 230-236) containing the catalytic nucleophile Asp-230 and two residues, Ala-231 and Lys-233, critical in substrate binding and reaction specificity [18;32]. While the guanidinium group of the arginine residue at position 239 may take the place of two water molecules, modelling shows that the space occupied by the aromatic side chain of tryptophan is not completely filled by arginine or leucine, thus reducing the structural stability of these variants (Fig. 3). Additionally, the W239R/L mutations also destroy a hydrogen-bonding interaction between the side chains of Asp-209 and Trp-239, compromising the stability of these variants even further. The creation of greater space within this region may also change the packing of a hydrophobic cavity close to the active site, altering the orientation of the catalytic residues for a more selective deprotonation of the acceptor saccharide than the water molecule. the active site induced by second shell mutants, (S77P, W239R/L) are essential for significant reduction in side reaction rates, with retention or even increase in primary catalytic function rates (Tables 2, 3 & 4) [33-36]. Effect of active site mutations on transglycosylation/hydrolysis activity -As the nature of the acceptor utilised determines reaction type, it seems logical that the residues at the acceptor subsites play a large role in determining reaction specificity. Previously it has been shown that the acceptor subsite residues of CGTases are important for the transglycosylation specificity of the enzyme [10;16]. Most mutations at the acceptor subsites, close to the catalytic nucleophile Asp-230 of Tabium CGTase, increased the hydrolytic activity significantly and lowered the transglycosylating activity of the enzyme (Fig. 6). To investigate whether mutations at the acceptor subsites, directly interacting with substrate, could enhance the transglycosylation specificity of CGTase, by lowering the competing hydrolysis reaction, at the +1 and +2

  new specialized function with partial retention of initial ancestral function as a side reaction [37], we propose that retention of hydrolysis by these enzymes is a remnant of ancestral function. The specialized glucanotransferases of GH13 most likely evolved from 'intermediate enzymes' similar to that of maltogenic amylase and neopullulanase. Both enzymes produce similar amounts of end products from both hydrolase and transglycosylase activity on starch and maltodextrin (Fig. 6) [38;39]. However, due to growing competition between microbes for available carbon source from starch and glucans, increased pressure was placed upon the evolution of these 'intermediate enzymes' toward transglycosylation activity, for selective uptake and storage of the available saccharide substrate. For instance, certain bacteria secrete CGTases to monopolise on the starch substrate, converting it into cyclodextrins which cannot be used by competing organisms [40;41]. Gradually these glucanotransferase enzymes became more specialized till hydrolysis decreased to a mere side reaction or disappeared completely. Branching enzymes of GH13 which catalyze the formation of -1,6 bonds, display no detectable hydrolytic activity on the amylose substrate [42]. These enzymes play a vital role in defining the structural and physical properties of the storage compound glycogen, in bacteria, archaea, fungi and animals. As all members of GH13, including amylases and transglycosylases, share both an identical catalytic machinery and mechanism, with highly similar active site architecture, the question remains as to how these from the recent elucidation of the induced-fit mechanism in the catalytic cycle of amylomaltase from GH77 [30]. As previously mentioned, short saccharide binding at the acceptor subsites during the final stages of catalysis is required for reorientation of active site residues to allow the acid/base catalyst side chain to attain a catalytically competent conformation. This ensures the preference for saccharide as acceptor over water for the transglycosylation reaction (Fig. 6). Mutations introducing this selective induced fit in GH13 glucanotransferases are most likely to have arisen on the outer regions of the enzymes active site. As our studies revealed, a single mutation on the outer regions of the active site is sufficient to induce subtle conformational changes to the catalytic residues resulting in increased preference for transglycosylation over hydrolysis activity. Introducing mutations neighbouring the catalytic residues may have too large a disruptive effect on the overall catalytic mechanism to increase primary catalytic function, as shown by investigation of our acceptor subsite library. To conclude, continuous enzyme evolution within the functionally diverse GH13 family has resulted in intermediate enzymes catalysing new function while partially retaining ancestral function as a side reaction. By applying directed evolution to our Tabium CGTase enzyme we have successfully lowered the side reactions of hydrolysis and coupling while retaining the primary cyclization activity. Both effective mutations generated, S77P and W239R were identified outside the active site region, indicating that subtle conformational changes are sufficient to retain primed preexisting catalytic function, while removing side reactions. † HL acknowledges financial support from the Netherlands Organization for Scientific Research (NWO). Biochemical Journal Immediate Publication. Published on 21 Apr 2008 as manuscript BJ20080353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080353 S t a g e 2 ( a ) P O S T -P R I N T TABLES & FIGURES

Biochemical

  Journal Immediate Publication. Published on 21 Apr 2008 as manuscript BJ20080353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080353 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

BiochemicalFigure 1 .

 1 Figure 1. Schematic representation of the four reactions catalysed by CGTase. A saccharide binds to the substrate binding subsites of CGTase followed by bond cleavage to yield an enzyme-glycosyl intermediate. The second step of the reaction involves the transfer of the covalently bound saccharide to an acceptor molecule. (A) In the primary cyclization reaction the OH-4 group of the non-reducing end saccharide is used as acceptor. (B) A short saccharide acts as acceptor for disproportionation. (C) For the side reaction of hydrolysis a water molecule replaces the saccharide as acceptor for shorter saccharide production. (D) Coupling, the reverse reaction of cyclization, involves binding of cyclodextrin, breakdown of ring structure, linearization of saccharide before subsequent breakdown of enzyme-glycosyl intermediate by attacking of an acceptor saccharide.

Figure 2 .

 2 Figure 2. Production of cyclodextrins and the short linear saccharides (glucose, maltose and maltotriose) in g/L from 10% (w/v) Paselli SA2 starch by wild-type and mutant CGTases at pH 6.0 and 60 °C. , and -cyclodextrins are indicated by black diamonds, circles, and squares, respectively, with short saccharides represented by open circles.

Figure 3 . 22 Biochemical

 322 Figure 3. Thermal denaturation curves of wild-type and mutant CGTases as measured with differential scanning calorimetry. The panel gives the apparent melting temperatures of variants.

Figure 4 .

 4 Figure 4. Structural representation of Tabium CGTase mutant, S77P, with heptasaccharide analog bound at the -4 to +3 subsites (Protein Data Bank 3BMW). Location of the S77P, W239, A231 and F260 residues within the protein structure are indicated by red spheres. The lower panel displays a close-up view of the catalytic core region from the above structure.

Figure 4

 4 Figure4was created using PyMOL[START_REF] Delano | The PyMOL Molecular Graphics System[END_REF].

Figure 5 .

 5 Figure 5. Molecular modelling of the location and effect of S77P mutation on the Tabium CGTase active site conformation. (A) Superimposition of the wild-type and S77P active sites.

Figure 6 . 6 , 24 Biochemical

 6624 Figure 6. Development of specialized enzyme function from a common evolutionary ancestor. (A) Both transglycosylation/hydrolase activities and ratios are shown for membersof family GH13. Also included are amylomaltases from GH77. 1, neopullulanase[START_REF] Kuriki | Controlling substrate preference and transglycosylation activity of neopullulanase by manipulating steric constraint and hydrophobicity in active center[END_REF]; 2, maltogenic amylase[START_REF] Cha | Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose[END_REF]; 3, Tabium CGTase; 4, amylosucrase[START_REF] Albenne | Molecular basis of the amylose-like polymer formation catalyzed by Neisseria polysaccharea amylosucrase[END_REF]; 5, CGTase mutant S77P; 6, Bacillus circulans 251 CGTase[START_REF] Kelly | Evolution toward small molecule inhibitor resistance affects native enzyme function and stability: generating acarbose insensitive cyclodextrin glucanotransferase variants[END_REF]; 7, maltosyltransferase [47]; 8, amylomaltase[START_REF] Kaper | Identification of acceptor substrate binding subsites +2 and +3 in the amylomaltase from Thermus thermophilus HB8[END_REF]; 9, amylomaltase [48]; 10, branching enzyme. Branching enzyme displays no detectable hydrolytic activity on the amylose substrate[START_REF] Takata | Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at alpha-(1-4)-and alpha-(1-6)-glucosidic linkages[END_REF]. (B) The effect of mutations in the donor and acceptor subsites of Tabium CGTase on the initial cyclization and hydrolytic rates with starch[10;16;20]. S A and S D indicate mutated residues located in or closest to the acceptor and donor subsites, respectively.

25 Biochemical

 25 Figure 2

Figure 3 26 BiochemicalF 6 Biochemical

 266 Figure 3

Table 1 .

 1 Data collection statistics and quality of the Thermoanaerobacterium

	thermosulfurigenes EM1 CGTase mutant S77P.
		S77P	S77P with inhibitor
	Data collection
	Spacegroup	P2 1 2 1 2 1	P2 1 2 1 2 1
	Cell axes a, b, c (Å)	73.6, 96.0, 114.2	73.8, 96.6, 115.2
	Resolution range (Å)	50.0 -1.6	50.0 -1.6
	Reflections total (unique)	353324 (95798)	384566 (102676)
	Completeness (%) a	89.9 (57.2)	94.2 (72.7)
	<I/ (I)> a	14.2 (5.2)	11.6 (3.6)
	R merge (%) a	7.6 (18.4)	10.0 (19.0)
	Refinement stastics
	No. of amino acids	683 (all)	683 (all)
	No. of Ca 2+ ions	2	2
	2-ions No. of Cl -ions No. of SO 4	5 -	3 1
	No. of glycerol molecules 4	7
	No. of water molecules	1023	1160
	Active site ligand	-	maltotriose-
			acarbose
	MBS3 ligand	-	maltose
	Final R-factor (%)	14.2	14.4
	Final free R-factor (%)	16.3	16.5

Root mean square deviation (rmsd) from ideal geometry

	Bond lengths (Å)	0.007	0.007
	Angles ( o )	1.1	1.1
	Ramachandran plot (%)
	favored	96.9	97.2
	allowed	3.1	2.8
	disallowed	0.0	0.0
	a Highest resolution shell in parentheses
		19

Table 2 .

 2 The -cyclization and hydrolysing activities of wild-type and mutant Tabium CGTases at 60 °C and pH 6.0.

	Enzyme	k cat (s -1 ) -cyclization Hydrolysis	Cyclization/ Hydrolysis	Fold-reduction a
	Wild-type	324 ± 6	40 ± 2	8.1	1
	S77P	182 ± 4	2.7 ± 0.3	67.4	15
	W239L	317 ± 0.1	29 ± 2	10.9	1.4
	W239R	247 ± 2	25 ± 3	9.9	1.6
	a Fold-reduction in hydrolytic activity of mutants compared to wild-type.

Table 3 .

 3 Kinetic parameters of the disproportionation reaction of wild-type and mutant Tabium CGTases at 60 °C and pH 6.0.

	Enzyme	k cat (s -1 )	K M, pNPG7 (mM) K M, maltose (mM) k cat /K M, pNPG7 (s -1 mM -1 )
	Wild-type	1244	0.15	3.3	8293
	S77P	558	0.24	2.4	2325
	W239L	1082	0.27	4.2	4007
	W239R	644	0.2	4.1	3220
			20

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Table 4 .

 4 Kinetic parameters of the coupling reaction of wild-type and S77P mutant Tabium CGTases at 60 °C and pH 6.0.

	Enzyme	k cat (s -1 )	K M (mM)	k cat / K M (s -1 mM -1 )
	-CD
	Wild-type	1408 ± 40	1.6 ± 0.2	880
	S77P	110 ± 3	1 ± 0.1	110
	W239L	1285 ± 49	1.8 ± 0.1	713
	W239R	644 ± 29	1.3 ± 0.2	495
	-CD
	Wild-type	285 ± 8	0.9 ± 0.07	317
	S77P	26 ± 1	0.7 ± 0.06	37
	W239L	295 ±10	0.8 ± 0.1	369
	W239R	160 ± 4	0.8 ± 0.1	200
	-CD
	Wild-type	321 ± 13	1.3 ± 0.2	247
	S77P	44 ± 3	1.7 ± 0.2	26
	W239L	472 ±19	1.8 ± 0.3	262
	W239R	254 ± 8	1.6 ± 0.2	159

Biochemical Journal Immediate Publication. Published on 21 Apr 2008 as manuscript BJ20080353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080353 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society


Carine M Bebrone

Christine Anne

Frédéric Kerff

Gianpero Garau

Kris De Vriendt

Raphaël Lantin

Bart Devreese

Jozef Van Beeumen

Otto Dideberg

J.-M Frère

Gianpiero Garau

Jean-Marie Frère

Moreno Galleni
email: mgalleni@ulg.ac.be.

Mutational analysis of the zinc and substrate binding sites in the

Keywords: EXAFS (Extended X-ray absorption fine structure), ICP-MS (inductively coupled plasma mass spectroscopy), PAC β-lactamase, carbapenems, zinc binding sites, substrate binding, sitedirected mutagenesis, enzymatic kinetics

Introduction

In vivo, class B β-lactamases [START_REF] Ambler | The structure of beta-lactamases[END_REF] require one or two zinc ions as enzymatic cofactors. By efficiently catalysing the hydrolysis of the β-lactam amide bond, these enzymes play a key role in bacterial resistance to this group of antibiotics. Several pathogens are now known to synthesize members of this class [START_REF] Rasmussen | Cloning and sequencing of the class B beta-lactamase gene (ccrA) from Bacteroides fragilis TAL3636[END_REF][START_REF] Watanabe | Transferable imipenem resistance in Pseudomonas aeruginosa[END_REF][START_REF] Massidda | The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-beta-lactamases[END_REF][START_REF] Osano | Molecular characterization of an enterobacterial metallo beta-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance[END_REF][START_REF] Rossolini | Characterization and sequence of the Chryseobacterium (Flavobacterium) meningosepticum carbapenemase: a new molecular class B beta-lactamase showing a broad substrate profile[END_REF][START_REF] Succi | Beta-lactamase genes of the penicillin-susceptible Bacillus anthracis Sterne strain[END_REF].

Metallo-β-lactamases exhibit very broad activity spectra [START_REF] Felici | An overview of the kinetic parameters of class B betalactamases[END_REF]. Moreover, they are not susceptible to the common β-lactamases inactivators [START_REF] Prosperi-Meys | Interaction between class B beta-lactamases and suicide substrates of active-site serine beta-lactamases[END_REF]. No clinically efficient inhibitor of metallo-β-lactamases has been found yet. The fact that some of the metallo-β-lactamases genes are plasmid-encoded represents an additional cause of concern [START_REF] Livermore | Carbapenemases: a problem in waiting?[END_REF][START_REF] Walsh | Metallo-βlactamases: the quiet before the storm[END_REF]. On the basis of the known sequences, three different lineages, identified as subclasses B1, B2, and B3, can be characterized [START_REF] Galleni | and the Metallo-β-lactamases Working Group (2001) Standard numbering scheme for class B beta-lactamases[END_REF][START_REF] Garau | Update of the standard numbering scheme for class B beta-lactamases[END_REF]. The enzymes exhibit two potential zinc binding sites containing His(Asn)116, His118 and His196 in subclasses B1 and B3 (B2) and Asp120, Cys221 (His121) and His263 in subclasses B1 and B2 (B3). Each subclass thus possesses a different set of potential zinc ligands.

The CphA metallo-β-lactamase produced by Aeromonas hydrophila belongs to subclass B2 which is characterized by a uniquely narrow specificity profile. In contrast to metallo-β-lactamases of subclasses B1 and B3, CphA efficiently hydrolyses only carbapenems and shows very poor activity against penicillins and cephalosporins [START_REF] Felici | An overview of the kinetic parameters of class B betalactamases[END_REF][START_REF] Segatore | High specificity of cphA-encoded metallo-beta-lactamase from Aeromonas hydrophila AE036 for carbapenems and its contribution to beta-lactam resistance[END_REF] and exhibits a maximum activity as a mono-zinc enzyme. The presence of a Zn 2+ ion in a second low affinity binding site noncompetitively inhibits the enzyme with a K i value of 46 µM at pH 6.5 [START_REF] Hernandez-Valladares | Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability[END_REF]. Recently, the structure of the mono-zinc CphA enzyme has been solved by X-ray cristallography [START_REF] Garau | A Metallo-beta-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem[END_REF]. Similarly to the known structures of metallo-β-lactamases of subclasses B1 [17][START_REF] Concha | Crystal structure of the wide-spectrum binuclear zinc beta-lactamase from Bacteroides fragilis[END_REF][START_REF] Carfi | 1.85 Å resolution structure of the zinc (II) beta-lactamase from Bacillus cereus[END_REF][START_REF] Fabiane | Crystal structure of the zinc-dependent beta-lactamase from Bacillus cereus at 1.9 Å resolution: binuclear active site with features of a mononuclear enzyme[END_REF][START_REF] Concha | Crystal structure of the IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor[END_REF][START_REF] Garcia-Saez | The 1.5 Å structure of Chryseobacterium meningosepticum zinc beta-lactamase in complex with the inhibitor, D-captopril[END_REF] and B3 [START_REF] Ullah | The crystal structure of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 Å resolution[END_REF][START_REF] Garcia-Saez | Three-dimensional structure of FEZ-1, a monomeric subclass B3 metallo-beta-lactamase from Fluoribacter gormanii, in native form and in complex with D-captopril[END_REF], the X-ray structure of CphA highlights an αββα sandwich with two central βsheets and α-helices on the external faces. The active site is located at the bottom of the β-sheet core. In contrast to all reported B1 and B3 subclass structures, a long α3 helix (Arg140-Leu161) is located near the active site groove of CphA. α3 is a key element of the "hydrophobic wall" which defines the active site pocket. Thus, whereas B1 and B3 enzymes exhibit a large cavity, the active site of CphA is very well defined and this explains the very narrow activity profile of the enzyme [START_REF] Garau | A Metallo-beta-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem[END_REF].

Contrary to the enzymes of subclasses B1 and B3, it was demonstrated that, in the subclass B2 CphA enzyme, a cysteine residue contributes to the first catalytic zinc binding site [START_REF] Garau | A Metallo-beta-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem[END_REF][START_REF] Hernandez-Valladares | Kinetic and spectroscopic characterization of native and metal-substituted beta-lactamase from Aeromonas hydrophila AE036[END_REF][START_REF] Heinz | Coordination geometries of metal ions in D-or L-captopril-inhibited metallo-beta-lactamases[END_REF][START_REF] Vanhove | Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase[END_REF]. This result suggested that the respective binding sites of the first and second zinc ions would be reversed in CphA as compared to B1 and B3 metallo-β-lactamases. However, on the basis of spectroscopic studies with another subclass B2 enzyme (ImiS), Crawford and his co-workers [START_REF] Crawford | Spectroscopic studies on cobalt(II)-substituted metallo-beta-lactamase ImiS from Aeromonas veronii bv[END_REF][START_REF] Costello | X-ray absorption spectroscopy of the zinc-binding sites in the class B2 metallo-betalactamase ImiS from Aeromonas veronii bv. sobria[END_REF] postulate that the second zinc binding site is not the traditional "histidine" site, but involves both His118 and Met146. Furthermore, by soaking CphA crystals in a biapenem solution, a reaction intermediate could be trapped into the active site of the enzyme [START_REF] Garau | A Metallo-beta-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem[END_REF]. The analysis of this structure allowed us to select several residues potentially important for substrate binding such as Val67, Thr157, Lys224 and Lys226, respectively (figure 1).

The side-chain of Val67 is in van der Waals contact with the 1β-methyl group of biapenem. Val67 is a residue of the L1 loop (Tyr60-Val67) [START_REF] Garau | Update of the standard numbering scheme for class B beta-lactamases[END_REF][START_REF] Garau | A Metallo-beta-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem[END_REF] and is part of the "hydrophobic wall" which defines the active site of CphA. The Val67 residue is conserved in B2 metallo-β-lactamases and in the BcII, CcrA and IMP-1 B1 enzymes [START_REF] Garau | Update of the standard numbering scheme for class B beta-lactamases[END_REF]. The hydroxyl group of Thr157 makes hydrogen bonds with the hydroxyethyl group on C6 of biapenem and, after hydrolysis, with the newly formed carboxylate group [START_REF] Garau | A Metallo-beta-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem[END_REF]. Thr157, a residue of the α3 helix located near the active site groove, is only conserved in subclass B2 enzymes. As in CcrA and IMP-1 subclass B1 enzymes [START_REF] Concha | Crystal structure of the IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor[END_REF][START_REF] Yang | Kinetic properties and metal content of the metallo-β-lactamase CcrA harboring selective amino acid substitutions[END_REF][START_REF] Yanchak | Mutational analysis of metallo-β-lactamase CcrA from Bacteroides fragilis[END_REF][START_REF] Haruta | Characterization of the active-site residues asparagine 167 and lysine 161 of the IMP-1 metallo-betalactamase[END_REF][START_REF] Materon | Analysis of the context dependent sequence requirements of active site residues in the metallo-βlactamase IMP-1[END_REF], the Lys224 side-chain forms an electrostatic interaction with the invariant carboxylate group on C3 of the β-lactam. It is conserved in B2 and B1 enzymes with the exception of VIM-2 [START_REF] Garau | Update of the standard numbering scheme for class B beta-lactamases[END_REF]. The Lys226 side chain is the only charged side chain near the C2 bicyclotriazoliumthio group of biapenem.

In order to evaluate their importance in metal binding and/or catalysis, substitutions of residues potentially involved in zinc binding have been performed by site-directed mutagenesis (H118A, D120A, D120T, H196A and H263A). To examine the effects of the charge or size of the residues on the substrate specificity, valine at position 67 was replaced by Ala, Ile and Asp, threonine at position 157 by Ala, lysine residues at positions 224 and 226 by Gln. Lys224 was also replaced by Gln in the N116H-N220G extended spectrum double mutant [START_REF] Bebrone | Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-β-lactamase by site-directed mutagenesis[END_REF].

Experimental Bacterial strains and vectors

Escherichia coli DH5α (Stratagene Inc., La Jolla, Calif.) and E. coli BL21 (DE3) pLysS Star (Invitrogen, Merelbeke, Belgium) were used as hosts for the construction of the different expression vectors and for the production of βlactamases, respectively. pET9a-CphA [START_REF] Hernandez-Valladares | Overproduction and purification of the Aeromonas hydrophila CphA metallo-betalactamase expressed in Escherichia coli[END_REF] was used as plasmid template in the PCR site-directed mutagenesis amplifications. pK18-CphA vector was constructed by a ligation of the EcoRI-SmaI fragment of pAS20R [START_REF] Massidda | The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-beta-lactamases[END_REF] and the SmaI-HindIII fragment of pET9a-CphA [START_REF] Hernandez-Valladares | Overproduction and purification of the Aeromonas hydrophila CphA metallo-betalactamase expressed in Escherichia coli[END_REF] in position EcoRI-HindIII of the polylinker of the pK18 vector [START_REF] Pridmore | New and versatile cloning vectors with kanamycinresistance marker[END_REF]. Moreover, a unique PstI restriction site was introduced by site-directed mutagenesis at the end of the signal peptide coding sequence. The CphAPstIa and CphAPstIb primers used for this purpose are described in the supplementary material section. pK18-CphA was then used as template in the pentapeptide scanning reaction [START_REF] Hallet | Pentapeptide scanning mutagenesis: random insertion of a variable five amino acid cassette in a target protein[END_REF] described below.

Chemicals and antibiotics

The primers used for the mutagenesis were synthesized by Eurogentec (Liège, Belgium). Kanamycin was purchased from Merck (Darmstadt, Germany). Imipenem (Δε 300 = -9000 M -1 cm -1 ) was from Merck Sharp and Dohme Research Laboratories (Rahway, NJ, USA), biapenem (Δε 294 = -9900 M -1 cm -1 ) from Shionogi Pharmaceutical Co., (Osaka, Japan), ampicillin, benzylpenicillin (Δε 235 = -775 M -1 cm -1 ), cephaloridine (Δε 260 = -10000 M -1 cm -1 ), cefotaxime (Δε 260 = -7500 M -1 cm -1 ) from Sigma (St Louis, Mo., USA) and nitrocefin (Δε 482 = -15000 M -1 cm -1 ) from Unipath Oxoid (Basingstoke, UK). The variations of the molar extinction coefficient upon β-lactam hydrolysis are from references [START_REF] Mercuri | Probing the specificity of the sub-class B3 FEZ-1 metallo-beta-lactamase by sitedirected mutagenesis[END_REF] (biapenem) and [START_REF] Matagne | The beta-lactamase cycle: a tale of selective pressure and bacterial ingenuity[END_REF]. The antibiotics used in this study are illustrated in figure 1 (biapenem) and in the supplementary material section.

Site-directed mutagenesis

The Quick Change Site-directed mutagenesis kit (Stratagene Inc., La Jolla, Calif.) was used to introduce all the mutations. The primers designed for these experiments are listed as supplementary material. The mutants were constructed using pET9a-CphA WT as template. The triple mutant N116H-N220G-K224Q was constructed using pET9a-N116H-N220G mutant [START_REF] Bebrone | Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-β-lactamase by site-directed mutagenesis[END_REF] as template. The introduction of the desired mutations and the absence of additional unwanted mutations were always verified by sequence analysis.

Pentapeptide scanning

A pentapeptide scanning reaction [START_REF] Hallet | Pentapeptide scanning mutagenesis: random insertion of a variable five amino acid cassette in a target protein[END_REF] was performed with the help of the GPS TM -LS Scanning System (New England Biolabs, Hitchin, UK). This system allows the insertion of 15 bp linkers at random positions throughout the blaCphA gene of the pK8-CphA plasmid. The different plasmids were used to transform E. coli DH5α by electroporation. The colonies were selected on LB-agar plates supplemented by 50 µg/ml kanamycin and 10 µg/ml imipenem, and 50 µg/ml kanamycin and 8 µg/ml benzylpenicillin, respectively. The plasmids were prepared and the different blaCphA genes sequenced to determine the positions of the insertion.

Protein expression and purification

The genes encoding the mutant proteins were cloned into pET9a using BamHI and NdeI restriction sites. The different vectors were then introduced into E. coli strain BL21 (DE3) pLysS Star. Overexpression and purification of mutant proteins were performed as described for the wild-type protein [START_REF] Hernandez-Valladares | Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability[END_REF][START_REF] Vanhove | Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase[END_REF][START_REF] Hernandez-Valladares | Overproduction and purification of the Aeromonas hydrophila CphA metallo-betalactamase expressed in Escherichia coli[END_REF] with the following modification. Bacteria expressing the wild-type protein were grown for 8 h at 37 °C in 2XYT medium while those expressing the mutants were grown for 24 h at 18 °C in the same medium. This temperature was made necessary by the fact that the mutants were produced mainly in a non-soluble form (inclusion bodies) when the cultures were grown at 37 °C.

ESI-MS and Metal Content Determination

Samples of wild-type and mutant CphA enzymes were equilibrated in 10 mM ammonium acetate buffer (pH 6.5) by centrifugal filtration prior to ESI-MS. For each protein mass spectra were obtained under both denaturing and native conditions. Denaturation of the protein was performed by dilution of the protein to a final concentration of 2-5 µM in a 50:50 (v/v) water/acetonitrile mixture containing 0.1% formic acid. The native protein was diluted to 10 -15 µM in 10 mM ammonium acetate.

All mass spectra were acquired on a Q-TOF1 mass spectrometer (Micromass, UK) equipped with a nano-ESI source using Au/Pd coated borosilicate needles purchased from Protana (Odense, Denmark). Capillary voltage was set at 1250 V and cone voltage at 40 V and 60 V for the denatured and native proteins respectively.

Acquisition time was 3 to 5 min across an m/z range of 400 -3000. The mass spectra were processed with MassLynx v3.1 software of Micromass. The instrument was calibrated using a mixture of myoglobin and trypsinogen. The zinc content of each protein was derived from the mass difference between the native and denatured proteins.

Determination of the zinc content using ICP/MS

Protein samples were dialyzed against 15 mM sodium cacodylate pH 6.5.

Protein concentrations were determined by measuring the absorbance at 280 nm (ε 280 = 38500 M -1 cm -1 ). Zinc contents were determined by Inductively Coupled Plasma Mass Spectroscopy (ICPMS) as previously described [START_REF] Hernandez-Valladares | Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability[END_REF][START_REF] Vanhove | Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase[END_REF].

Stability toward chaotropic agents

The stability of the different proteins was studied by fluorescence. The wildtype and mutant enzymes (0.05 mg/ml) were incubated for 16h in the presence of increasing urea concentrations (0-8M) at 4°C. Fluorescence emission spectra of the

Circular dichroism

Circular dichroism spectra of the different enzymes (0.5 mg/ml) were obtained using a JASCO J-810 spectropolarimeter. The spectra were scanned at 20°C with 1 nm steps from 200 to 250 nm (far UV) and from 250 to 310 nm (near UV).

Determination of kinetic parameters

The hydrolysis of the antibiotics was monitored by following the absorbance variation resulting from the opening of the β-lactam ring, using an Uvikon 860 spectrophotometer equipped with thermostatically controlled cells and connected to a microcomputer. For biapenem, imipenem, benzylpenicillin, nitrocefin, cephaloridine and cefotaxime, the wavelengths were 294, 300, 235, 482, 260 and 260 nm, respectively. Cells with 0.2 -1.0 cm pathlengths were used, depending on the substrate concentrations. When the K m values of the studied enzymes were sufficiently high, the k cat and K m parameters were determined either under initial-rate conditions, using the Hanes linearization of the Henri-Michaelis-Menten equation, or by analysing the complete hydrolysis time courses [START_REF] De Meester | Automated analysis of enzyme inactivation phenomena. Application to beta-lactamases and DD-peptidases[END_REF]. Low and very high K m values were determined as K i 's using imipenem or nitrocefin as reporter substrates. In the cases of low K m values, the k cat values were obtained from the initial hydrolysis rates measured at saturating substrate concentration, and in the cases of high K m values, k cat was derived from the k cat /K m ratio. All experiments were performed at 30° C in 15mM sodium cacodylate buffer pH 6.5.

Inactivation by EDTA

Inactivation of CphA and the H118A, H196A, H263A and D120A mutants by EDTA was studied in 15 mM sodium cacodylate, pH 6.5 at 30°C, using 200 µM imipenem as the reporter substrate and in the presence of different concentrations of EDTA in a total volume of 0.5 ml. Since the inactivation time courses followed pseudo-first-order kinetics, the rate constants characterizing the inactivation of the enzyme were calculated from the dependence of the pseudo-first-order constant k i upon the chelating agent concentration on the basis of the following model:

E.Zn + C E.Zn.C E + Zn.C K k +2 k -2
in which E.Zn, C, E.Zn.C, E, and Zn.C are the metalloenzyme, the chelator, a ternary metalloenzyme-chelator complex, the apoenzyme, and the metal-chelator complex, respectively. K is the dissociation constant of the E.Zn.C ternary complex, k +2 the individual rate constant for the dissociation of E.Zn.C into E + Zn.C and and k -2 the association constant, which was negligible in the case of the H263A mutant.

As [Zn.C] is higher than [E], the ternary complex formation from the apoenzyme and the zinc-chelatant complex is characterized by a pseudo-first-order constant:

k -2 ' = k -2 [Zn.C] [ ] [ ] ( ) [ ] [ ] 9 2' - 2 i k C k k + + + = + (Eq. 1)
The individual values of K and k +2 were determined by fitting the values of k i to

equation 1 m m C / S K K K
where [S] and K m were the concentration and the K m value of the reporter substrate, respectively.

Enzymatic measurement in the presence of increasing concentrations of zinc and determination of K D2

Residual activity in the presence of increasing concentrations of zinc was measured at 30°C in 15mM sodium cacodylate pH 6.5 as already described [START_REF] Vanhove | Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase[END_REF].When binding of the second zinc ion resulted in a complete loss of activity, the data were analysed according to Eq.2 [START_REF] Vanhove | Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase[END_REF]:

RA = [K D2 / ([Zn] + K D2 )] x 100 (Eq. 2)
where RA is the residual activity and K D2 is the dissociation constant for the second zinc ion.

Results and discussion

A mutant that presents a Leu-Phe-Lys-His-Val insertion located just after Val67 (V67 LFKHV ) was obtained by a pentapeptide scanning experiment. The E. coli DH5α cells transformed by pK18-CphA V67 LFKHV were unable to grow on LB-agar plates supplemented by 10 µg/ml imipenem, in contrast to bacteria producing the wild-type enzyme. These cells were also unable to grow in the presence of 8 µg/ml ampicillin. The V67 LFKHV mutant was produced in the periplasmic fraction of E. coli DH5α (result not shown).

The different mutants were expressed in E. coli BL21 (DE3) pLysS Star and purified to homogeneity. The presence of the mutations was verified by mass spectrometry (table 1). Within experimental errors, the mutants were found to exhibit the expected masses, with the exception of V67 LFKHV mutant which presented a mass deficit of 92 Da. The N-terminal sequence of this mutant showed that there is no amino acid loss at the N-terminal side. The difference between the calculated and measured masses could thus be due to the loss of the C-terminal serine.

The stability of the different mutants against urea as denaturating agent was similar to that of the wild-type enzyme (C 1/2 = 2.7 -3 M) except for T157A (C 1/2 = 2.2 M) and H263A (C 1/2 = 2.4 M) mutants which turned out to be a little less stable.

All the mutants had far-UV CD spectra superimposable on that of wild-type CphA (not shown). The tertiary structures were conserved for H118A and H196A

while the environment of one or more aromatic residues was modified in H263A, D120A and D120T when compared to the wild-type enzyme (supplementary material). Near-UV CD spectra and fluorescence emission spectra of the N116H-N220G-K224Q and V67 LFKHV mutants suggested a small conformation change in the protein tertiary structure (not shown). Such a small conformation change had already been observed for the N116H-N220G double mutant [START_REF] Bebrone | Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-β-lactamase by site-directed mutagenesis[END_REF].

The activities of the mutants were measured against several compounds representing the three major families of β-lactam antibiotics, namely carbapenems, penicillins and cephalosporins, in the absence of added zinc (free zinc concentrations below 0.4 µM) (tables 3 and 5). Under these conditions, all the mutants, except ). V67 LFKHV already exhibited a di-zinc form (30 %) in conditions where the wild-type enzyme is only mono-zinc (table 1).

I. Mutational analysis of zinc binding sites

Although His118 and His196 are not involved in the binding of the catalytic metal ion [START_REF] Garau | A Metallo-beta-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem[END_REF], their replacement by Ala drastically reduces the enzymatic activity, suggesting important roles for these residues in the catalytic mechanism. The H118A and H196A mutations lead to an at least 1000-fold decrease in the activity against carbapenems compared to the wild-type enzyme (table 3). This result is perfectly in agreement with the structural data. Garau et al. suggested that His118 is the general base which activates the hydrolytic water molecule and that His196 contributes to the oxyanion hole by forming a hydrogen bond with the carbonyl oxygen of the β-lactam bond. Moreover, these residues might stabilize the carboxylate which is formed upon hydrolysis of the substrate [START_REF] Garau | A Metallo-beta-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem[END_REF]. This "active role" of His118 and His196 contrasts with the results obtained for Asn116, which in subclass B2 enzymes replaces His116, one of the zinc ligands in B1 and B3 enzymes. Indeed, whereas the His116Ser replacement reduces the activity of the B1 Bacillus cereus enzyme 15-20 fold, the Asn116Ser replacement in CphA yielded an enzyme whose catalytic and zinc-binding properties were similar to those of the wild-type enzyme. These results indicate that Asn116 in B2 enzymes is not a "functional substitute" of His116 in B1 and B3 [START_REF] Vanhove | Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase[END_REF].

After replacement of Asp120 and His263 by residues which cannot serve as zinc ligands, the mutant proteins could still bind one zinc ion but are also catalytically impaired in a dramatic manner. The D120A, D120T and H263A mutations result in a very large decrease in the activities against carbapenems (at least 50 000 fold) mainly due to strongly decreased k cat values (table 3). This very low residual activity could be explained by a poor positioning of the Zn 2+ ion in the active site of the mutants and a deformation of its coordination sphere. In this respect, it is interesting to note that the Asn220Gly mutation, which does not directly affect any of the zinc ligands but is situated next to Cys221, causes a displacement of the latter in 20 % of the molecules The effect of the zinc ion concentration on the activity has also been studied.

The binding of the second zinc ion is severely affected by the His118Ala and His196Ala mutations (figure 2a). From these curves, the H118A and H196A mutants are found to exhibit much higher K D2 values (940 ± 80 and 1250 ± 50 µM respectively) than the wild-type enzyme (46 ± 3 µM ). The poorer binding of the second zinc by CphA H118A was also demonstrated by mass spectrometry, which failed to detect the di-zinc form of this mutant [START_REF] De Vriendt | Monitoring the Zinc Affinity of the Metallo-beta-Lactamase CphA by Automated nanoESI-MS[END_REF]. This indicates that both histidine residues are involved in the binding of the second, inhibitory, zinc ion.

This interpretation is in disagreement with the conclusions of Crawford et al. [START_REF] Crawford | Spectroscopic studies on cobalt(II)-substituted metallo-beta-lactamase ImiS from Aeromonas veronii bv[END_REF] that the inhibitory zinc binds at a distance of at least 0.5 nm from the catalytic one in the ImiS B2 enzyme. Our results could be reconciled with this hypothesis if both our mutations resulted in significant conformational changes. On the basis of CD data (supplementary material), this does not seem to be the case. According to the structure, the presence of a second zinc ion in the active site interacting with His118 and His196 would not require any major rearrangement.

There is indeed a free space between those residues and Cys221, Asp120 and Asn116.

This second zinc ion would approximately be located at a position similar to that of the first zinc ion in the subclasses B1 and B3 β-lactamases and two of the zinc ligands would be conserved. Compared to the available structure of CphA, the only modification needed would be a 180° rotation around the Cβ-Cγ bond of the His196 side chain to bring the Nε2 atom in the right orientation (figures 3a and 3b).

From this crude modelling, the identity of the third ligand of the second zinc ion is not clear. However, the fact that the Cys221 CphA mutants no longer bind zinc [START_REF] Vanhove | Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase[END_REF] while mutants of the two other ligands (Asp120 and His263) of the first zinc binding site retain relatively high affinities for zinc (K D < 0.4 µM) points towards a role of Cys221 in the binding of both zinc ions. The role of a cysteine in the simultaneous binding of two zinc ions is a known feature that has for example been observed in the structure of the DNA-binding domain of the Gal4 transcription factor [START_REF] Baleja | Refined solution structure of the DNA-binding domain of GAL4 and use of 3J(113Cd,1H) in structure determination[END_REF]. The second zinc binding site described here would likely be more energetically favourable (one side chain rotation) than the major rearrangement required to organise a zinc binding site involving the embedded Met146. The presence of a sulphur in the second binding site that lead to the identification Met146 could maybe be explained by the involvement of Cys221 in the binding of both ions.

The affinities of the Asp120 and His263 mutants are nonetheless probably decreased when compared to that of the wild-type enzyme as indicated by the higher rates of inactivation by EDTA (table 4). They are probably between those observed for site 1 and site 2 in the wild-type enzyme for which the K D1 is below 10 pM [START_REF] Wommer | Substrateactivated zinc binding of metallo-beta-lactamases: physiological importance of mononuclear enzymes[END_REF] and K D2 is 46 µM [START_REF] Hernandez-Valladares | Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability[END_REF]. Because the residual activity of these mutants can be inhibited by EDTA and because the K D2 's deduced from the inhibition by zinc are still relatively close to 46 µM (figure 2b) (K D2 values are 15 ± 1.5 µM for H263A and 44 ± 6 and 41 ± 2 µM for D120A and D120T respectively), the binding of the first zinc to these mutants might result from a reduced affinity for site 1 rather than an improved affinity for the inhibitory site 2. The occupation of both sites at the same time has not been observed [START_REF] De Vriendt | Monitoring the Zinc Affinity of the Metallo-beta-Lactamase CphA by Automated nanoESI-MS[END_REF].

Mutational analysis of putative substrate binding site residues

Our kinetic results are in good agreement with the crystallographic structure of CphA in complex with biapenem [START_REF] Garau | A Metallo-beta-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem[END_REF] and confirm the importance of the Val67 residue for the binding of this substrate into the active site of CphA. The K m values of V67I and V67D for biapenem strongly increase, so that only k cat /K m could be measured (table 5). This phenomenon could be due to a less favourable interaction with the 1-β-methyl group of the antibiotic. In addition, we show that a hydrophobic interaction between Val67 and the C2 methyl groups of penicillin is likely, since the K m value of V67D for benzylpenicillin is larger than that of the wild-type CphA enzyme (table 5). A similar interaction has already been proposed between the BcII subclass B1 enzyme and benzylpenicillin [START_REF] Prosperi-Meys | Active-site mutants of class B β-lactamases: substrate binding and mechanistic study[END_REF]. Moreover, Simona et al. have recently suggested a possible role of the L1 loop (Tyr60-Val67) for the mechanism of binding carbapenem molecules [START_REF] Simona | Protonation state and substrate binding to B2 metallo-β-lactamase CphA from Aeromonas hydrophila[END_REF].

As for the wild-type CphA [START_REF] Hernandez-Valladares | Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability[END_REF], the binding of a second zinc ion inhibits in a noncompetitive manner the hydrolysis of imipenem by the Val67 mutants. The dissociation constants for the second zinc ion are similar to that of wild-type enzyme for V67I and V67D (46 ± 3 µM) or slightly reduced for V67A (16 ± 3 µM).

The V67 LFKHV mutant, with a five-residue insertion just after Val67, presents a strongly decreased affinity for biapenem, imipenem and nitrocefin (table 5). The extended loop might fold above the active site groove and in this way reduce the active site accessibility. The lengthening of the L1 loop and/or the introduction of a charged residue (Lys) could have a negative effect on the affinity of the enzyme for its substrate. The apparent dissociation constant for the second zinc ion is below 1 µM whereas it is 46 µM for the wild-type CphA. Together with the fact that this mutant exhibits a di-zinc form (30 %) in conditions where the wild-type enzyme is only mono-zinc, this result suggests that the His residue in the inserted pentapeptide might probably interact with the second zinc ion, thus recreating a possible second "3 His" zinc binding site (His67 d -His118-His196). However, a significant rearrangement would be necessary in order to bring the additional histidine into sufficiently close proximity to His118 and His196. Consequently, this hypothesis should be confirmed by EXAFS and PAC experiments and by the resolution of the 3D structure.

Various modifications can broaden the activity spectrum of the strict CphA carbapenemase. The N116H-N220G extended-spectrum mutant was obtained by a site-directed approach on the basis of sequence alignments between members of the B2 and B1 subclasses [START_REF] Bebrone | Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-β-lactamase by site-directed mutagenesis[END_REF]. In the present study, surprisingly, the replacement of Thr157 by an alanine results in a significant modification of the activity spectrum of the enzyme, since the catalytic efficiency of this mutant versus benzylpenicillin, cefotaxime and nitrocefin is larger than that of the wild-type enzyme. However, although T157A is far from being as efficient as the N116H-N220G mutant [START_REF] Bebrone | Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-β-lactamase by site-directed mutagenesis[END_REF], it retains, in contrast, a good activity versus imipenem (table 5).

Thr157 does not seem to play an important role in the CphA catalytic mechanism, but according to the structure of CphA in complex with modified biapenem (PDB code 1X8I) and the proposed mechanism for enzymatic activity on carbapenems [START_REF] Garau | A Metallo-beta-lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and its Complex with Biapenem[END_REF], the C6 (C7 for cephalosporins) side chains of β-lactam antibiotics are likely located in the environment of Thr157 during hydrolysis. A residue with a slightly reduced bulkiness could therefore be more suited to accommodate the larger and differently oriented side chain of most β-lactam antibiotics compared to carbapenems. This could explain the more extended spectrum observed for this mutant. Because the T157A mutant only retains a good activity against imipenem while the interactions of imipenem and biapenem with Thr157 should be affected similarly, we believe that this residue is more important for the optimum positioning of the substrate than for its affinity for the active site. The bulkier and more rigid biapenem would therefore need an extra anchoring to be perfectly oriented while imipenem would fit more naturally into the active site.

Zinc ions inhibit the hydrolysis of imipenem and benzylpenicillin by T157A in a noncompetitive manner, but the di-zinc form of this mutant retains 40 % of activity versus benzylpenicillin while it is practically completely deprived of activity versus imipenem (figure 4a). The dissociation constants for the second zinc ion are similar for both substrates and similar to that of the wild-type enzyme (respectively 46 ± 3 µM for T157A and imipenem, 55 ± 11 µM for T157A and benzylpenicillin and 46 ± 3 µM for the wild-type enzyme and imipenem [START_REF] Hernandez-Valladares | Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability[END_REF]). In contrast to the N116H-N220G mutant [START_REF] Bebrone | Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-β-lactamase by site-directed mutagenesis[END_REF], imipenem and benzylpenicillin seem in this case to be hydrolyzed via the same mechanism. The activity of the wild-type CphA against benzylpenicillin is too Our kinetic results show that Lys224 is important for the binding of β-lactam antibiotics into the active site of CphA, as already observed for the subclass B1

enzymes CcrA and IMP-1 [START_REF] Concha | Crystal structure of the IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor[END_REF][START_REF] Yang | Kinetic properties and metal content of the metallo-β-lactamase CcrA harboring selective amino acid substitutions[END_REF][START_REF] Yanchak | Mutational analysis of metallo-β-lactamase CcrA from Bacteroides fragilis[END_REF][START_REF] Haruta | Characterization of the active-site residues asparagine 167 and lysine 161 of the IMP-1 metallo-betalactamase[END_REF][START_REF] Materon | Analysis of the context dependent sequence requirements of active site residues in the metallo-βlactamase IMP-1[END_REF]. The K224Q mutant exhibits a decreased catalytic efficiency versus carbapenem substrates when compared to the wild-type enzyme. The individual k cat and K m values could not be determined. In the same way, the K m value for benzylpenicillin strongly increases (table 5). This role is in good agreement with the crystallographic structure of CphA in complex with biapenem which shows an electrostatic interaction between Lys224 and the invariant C3 carboxylate of the β-lactam antibiotic and with the simulations of Xu and his coworkers [START_REF] Xu | Catalytic mechanism of class B2 metallo-βlactamase[END_REF].

The N116H-N220G-K224Q triple mutant presents increased K m values and decreased k cat values when compared to the N116H-N220G double mutant (table 5).

This triple mutant, while significantly less active than the N116H-N220G double mutant, retains a relatively extended spectrum when compared to the wild-type enzyme. It is particularly interesting since, as already observed for the N116H-R121H-N220G triple mutant [START_REF] Bebrone | Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-β-lactamase by site-directed mutagenesis[END_REF] and in contrast to the wild-type enzyme, added zinc behaves as an activator. The hydrolysis rate of imipenem and nitrocefin by the N116H-N220G-K224Q triple mutant strikingly increases 1.6 and 2.5-fold at saturating Zn 2+ concentration (figure 4b). The dissociation constant for the second zinc ion determined for this triple mutant (4.0 ± 1.1 µM and 1.6 ± 0.2 µM with imipenem and nitrocefin respectively as substrates) is similar to that of the N116H-N220G double mutant (5.0 ± 0.9 µM [START_REF] Bebrone | Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-β-lactamase by site-directed mutagenesis[END_REF]). For all the other mutants, the hydrolysis of nitrocefin is independent of zinc concentration.

This phenomenon remains difficult to explain in the absence of structural data on the di-zinc inhibited form of the wild-type enzyme but it is quite likely that binding of the second zinc ion induces distinct structural changes in the wild-type and mutant proteins.

The replacement of Lys226 only affects the kinetic parameters of biapenem.

Although the k cat value is increased, the catalytic efficiency remains similar to that of the wild-type enzyme. The K226Q mutation does not significantly modify the affinity for the second zinc ion (K D2 = 85 ± 10 µM). All experiments were performed at 30°C in 15 mM sodium cacodylate pH 6.5. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Legends to figures

6

  Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T

7

  Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T enzymes were recorded at 20°C with a Perkin Elmer LS50B luminescence spectrometer using excitation and emission wavelengths of 280 and 333 nm, respectively.

8

  Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T When binding of the second zinc ion resulted in an incomplete loss of activity or in an increase of activity, Eq.3 was used [27]: RA = [(K D2 +α[Zn])/([Zn]+K D2 )] x 100 (Eq. 3) where α represents the ratio of activity at saturating zinc concentration versus activity in the absence of added zinc (Act. [Zn] (∞) / Act. [Zn] (0) ). Experimental data were fitted to Eq.2 or 3 by non-linear regression analysis with the help of the Sigma Plot software. 10 Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T

  V67 LFKHV , were shown to contain one zinc ion, as does the wild-type enzyme (tables 1 11 Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T and 2

[16]. 12 Biochemical

 12 Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N TICP/MS shows that, in contrast to the wild-type enzyme, these five mutant enzymes remain in their mono-zinc forms even in the presence of 100 µM free zinc (table2).

  Costello et al. have shown that the inhibitory zinc is bound by a sulphur[START_REF] Costello | X-ray absorption spectroscopy of the zinc-binding sites in the class B2 metallo-betalactamase ImiS from Aeromonas veronii bv. sobria[END_REF]. The sulphur ligand in the inhibitory site should come from a methionine residue, since the only cysteine residue (Cys221) is a ligand of the first zinc ion. The mutation of Met146 to isoleucine abolishes metal inhibition. However, mutations of other CphA residues, which have not been implicated as ligands of the second zinc ion, can have similar effects (lower inhibition or even activation by excess of zinc, as observed in the part "Mutational analysis of putative substrate binding site residues" of the present work). Moreover, a careful examination of the CphA 3D structure indicates that His118 and Met146 are poorly positioned to bind the same zinc ion. The distance between their Cα's is 7.57 Å, while the distances between the sulphur of Met146 and the nitrogens of the imidazole group of His118 are 10.40 and 10.91Å respectively. Also, the space between these residues does not allow the accommodation of a zinc ion because it is occupied by the backbone of the Asn114-His118 loop connecting strand β6 and helix α2.Moreover, an involvement of His118 and His196 in the binding of the inhibitory zinc ion makes sense in the light of the strongly decreased activity of the mutants (table3) 13 Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T since this binding would prevent them from playing their catalytic roles in the hydrolysis of carbapenems.

14 Biochemical

 14 Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T II.

16 Biochemical

 16 Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T low to allow meaningful measurements, however zinc also behaves as an inhibitor in this case.

Biochemical

  Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T Conclusion Our results indicate that His118 and His196 participate in the catalytic mechanism of the subclass B2 CphA metallo-β-lactamase for which an intact Asp120-Cys221-His263 site is essential. The position of the second inhibitory zinc ion is not identified with certainty, but it is likely equivalent to the "histidine" site observed in subclasses B1 and B3 enzymes. His118 and His 196 are involved in the binding of this second zinc and Cys221 is the leading candidate as the third ligand but the geometry is maybe not optimum and Asn116 or Asp120 could play a role too. The present study underlines the importance of Val67, Thr157 and Lys224 residues for the carbapenem binding into the active site of CphA. Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

29 Biochemical

 29 Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1 a

 1 Figure 1 a) Structure of biapenem b) Structure of the reaction intermediate c) View of the active site of CphA in complex with the reaction intermediate.

Figure 2 Residual

 2 Figure 2 Residual activity measured in the presence of increasing concentrations of zinc. a) WT, H118A, H196A and imipenem. The curves are the best fits using Eq.2. (WT: solid, H118A: dashed, H196A: dotted). b) WT, D120A, D120T, H263A and imipenem. The curves are the best fits using Eq.2. (WT: solid, D120A: dashed, D120T: dotted, H263A: dash-dot). All experiments were performed at 30°C in 15 mM sodium cacodylate pH 6.5. The substrate concentration was 100 µM. S.D. values are below 10 %.

Figure 3 Figure 4 Residual

 34 Figure 3 Representation of the active site of the CphA enzyme a) Structure of the mono-zinc active site PDB code 1X8G b) Model of a di-zinc active site with addition of a Zn ion and rotation of His196 side chain as the only modification when compared to a), the dashed lines show bonds to atoms within 3 Å of Zn2

FiguresFigure 1 () 35 Biochemical

 135 Figures

Figure 2 ( 36 Biochemical

 236 Figure 2 (Bebrone et al.)

Figure 3 (

 3 Figure 3 (Bebrone et al.)

Figure 4 ( 38 Biochemical

 438 Figure 4 (Bebrone et al.)

Table 1 . Masses and metal binding for WT CphA and mutants.

 1 The value between round brackets is obtained by assuming the elimination of the C-terminal serine. a The average mass of Zn ++ is 65.4. However, two or more protons can be displaced upon protein binding, giving the bound Zn ++ an apparent mass of 63.4.

	Tables

Table 1 (continued) 27

 1 Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T

Table 2 . Summary of zinc binding for wild-type CphA and the H118A, H196A, H263A, D120A, D120T mutants.

 2

	SD values were below 10%. N.D.: not determined.
	Protein	[Zn 2+ ] ≤ 0.4 µM		[Zn 2+ ] = 100 µM
		MS	ICP-MS	MS	ICP-MS
	WT	1	1.0	2	1.9
	H118A	1	0.9	N.D.	1.2
	H196A	1	N.D.	N.D.	1.2
	H263A	1	1.0	N.D.	0.9
	D120A	1	1.3	N.D.	1.3
	D120T	1	1.3	N.D.	1.3

Table 3 . Kinetic parameters of the wild-type CphA enzyme and the H118A, H196A, H263A, D120A and D120T mutants.

 3 Measurements were done at 30 °C in 15 mM sodium cacodylate pH 6.5. SD values were below 10%. N.D.: not determined. Values for the wildtype enzyme are fromVanhove et al., 2003 [27].

	Enzyme	Substrate	k cat (s -1 )	K m (µM)	k cat /K m (M -1 s -1 )
		Imipenem	1200	340	3 500 000
		Biapenem	300	170	1 800 000
	WT	Benzylpenicillin	0.03	870	35
		Nitrocefin	0.008	1300	6
		Cephaloridine	< 0.006	6000	< 1
		Imipenem	2.4	1150	2100
		Biapenem	0.1	730	150
	H118A	Benzylpenicillin	0.007	210	33
		Nitrocefin	0.005	30	170
		Cephaloridine	> 0.01	> 4500	2.2
		Imipenem	16
		Biapenem	N.D.

Table 4 . Individual parameters for the inactivation of WT, H118A, H196A, H263A and D120A CphA by EDTA.

 4

	SD values were below 10%.
		K (mM)	k +2 (s -1 )	k -2 (s -1 )	k +2 /K (M -1 s -1 )
	WT	5.5	0.007	0.0014	1.3
	H118A	5.2	0.002	0.003	0.4
	H196A	16.4	0.008	0.005	0.5
	H263A	0.01	0.005	/	500
	D120A	0.5	0.003	0.00001	6

30 Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 5 . Kinetic parameters of the WT, V67A, V67I, V67D, V67 LFKHV , T157A, K224Q, K226Q, N116H-N220G and N116H-N220G-K224Q CphA enzymes.

 5 Measurements were performed at 30°C in 15 mM sodium cacodylate pH 6.5. S.D. values are below 10 %. Values for the wild-type enzyme are fromVanhove et al., 2003 [27] and values for the N116H-N220G double mutant are fromBebrone et al., 2005 [34]. Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375

	Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375
	Enzyme	Antibiotics	k cat (s -1 )	K m (µM)	k cat /K m (M -1 s -1 )
	WT	Imipenem	1200	340 3 500 000
		Biapenem	300	170 1 800 000
	V67A V67I V67D	Benzylpenicillin Nitrocefin Cefotaxime Imipenem Biapenem Benzylpenicillin 0.004 0.03 0.008 > 0.0002 > 100 870 1300 1000 220 4 500 000 35 6 2 100 100 1 000 000 1000 4 Nitrocefin 0.005 26 180 Imipenem 1770 180 9 000 000 Biapenem > 50 > 1500 33 000 Benzylpenicillin 0.08 730 110 Nitrocefin 0.004 50 80 Imipenem 1280 310 4 000 000 Biapenem > 100 > 1500 66 000 Benzylpenicillin > 0.03 > 5000 6	THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375
		Nitrocefin	0.004	36	110
	V67 LFKHV	Imipenem	> 30	> 1 500 20 000
		Biapenem	> 2.4	> 1500	1 600
		Nitrocefin	> 0.006 > 2 000	3
	T157A	Imipenem	1680	570 3 000 000
		Biapenem	>130	> 1500 87 000
		Benzylpenicillin	1	550	1950
			31

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 23 May 2008 as manuscript BJ20080375 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080375 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

This work was supported by the Belgian Federal Government (PAI P5/33), grants from the FNRS (Brussels, Belgium, FRFC grants n° 2 4508.01, 2.4.524.03 and Lot. Nat. 9.4538.03) and an European Research Training Network (MEBEL contract HPTR-CT-2002-00264). The purchase of a Jasco J-810 spectropolarimeter was supported in part by a grant from the FRFC (contract number 2.4545.01). C.B. and F.K. were FNRS post-doctoral researchers. C.A. was the recipient of a Marie Curie fellowship.