1 Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, Policlinico Le Scotte, Viale Bracci, I-53100 Siena; 3 Dipartimento di Scienze e Tecnologie Biomediche Cattedra di Biochimica Clinica, Università di LAquila, Via Vetoio, Loc. Coppito, I-67100 LAquila, Italy; 2 Laboratoire dEnzymologie & Centre dIngénierie des Protéines, Institut de Chimie, Université de Liège, Bat. B6 Allée de la Chimie, Sart Tilman, B-4000 Liège, Belgium
Received 21 January 2002; returned 5 July 2002; revised 1 August 2002; accepted 28 October 2002
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Abstract |
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Introduction |
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During the last decade, two types of acquired metallo-ß-lactamases have been detected in Gram-negative pathogens, namely the IMP- and VIM-type enzymes. The IMP-type enzymes were detected in the early 1990s8,9 and have been the subject of several biochemical and structural investigations.7,915 The VIM-type enzymes were identified more recently,16,17 and considerably less is known about their functional and structural properties.17,18
The VIM-type enzymes belong to subclass B1 of molecular class B and include at least three variants: VIM-1, VIM-2 and VIM-3.16,17,19 Among them, VIM-2 is apparently the most widespread (it has been detected in clinical isolates from various European countries and the Far East),17,1921 whereas VIM-1 and VIM-3 producers encountered so far were isolated in Italy16 and Taiwan,19 respectively. The amino acid sequences of VIM-1 and VIM-2 diverge by 7%16,17 whereas VIM-3 differs from VIM-2 by only two residues.19 Notwithstanding their close structural similarity, notable differences have been reported for the kinetic parameters of VIM-1 and VIM-2 with some substrates,17,18 suggesting that the few structural differences between these two natural variants have a functional significance. However, in those studies, the experimental conditions used for kinetic measurements were not the same.17,18
In this work an expression system for high-level production of the VIM-2 enzyme in Escherichia coli was developed, and biophysical and biochemical characterizations of the purified VIM-2 enzyme were carried out under conditions identical to those previously adopted for VIM-1.18 Significant differences in the behaviour of the two enzymes with various substrates were confirmed, and molecular modelling was used to investigate the potential correlations between structural differences and the different enzyme kinetics.
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Materials and methods |
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E. coli XL-1 blue (Stratagene Inc., La Jolla, CA, USA) was routinely used for molecular cloning and plasmid propagation. E. coli BL21(DE3) (Novagen Inc., Madison, WI, USA) was used for metallo-ß-lactamase gene expression. Bacteria were grown in LuriaBertani (LB) medium,22 or in Buffered Super Broth medium (BSB: yeast extract, 20 g/L; tryptone, 35 g/L; NaCl, 5 g/L; buffered with 50 mM sodium phosphate buffer pH 7.0) supplemented with the appropriate antibiotic. Media components were from Difco Laboratories (Detroit, MI, USA).
Construction of the expression system for overproduction of VIM-2 in E. coli
The blaVIM-2 gene was amplified by PCR with primers VIM- 2-Fwd (5'-GGAATTCCATATGTTCAAACTTTTGAGTAAG), which added EcoRI (bold) and NdeI (underlined) restriction sites at the 5'-end of the gene, and VIM-2-Rev (5'-CCGGATCCTGCTACTCAACGACTG), which added a BamHI restriction site (bold) after the blaVIM-2 stop codon. Amplification was carried out in 50 µL using 50 pmol of each primer and the Expand PCR system (Roche Biochemicals, Mannheim, Germany), under the conditions recommended by the manufacturer, and the following cycling parameters: initial denaturation at 94°C for 3 min; denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 1 min, repeated for 30 cycles; final extension step at 72°C for 10 min. Plasmid pVRP193 (50 ng)21 was used as template. The resulting 0.8 kb amplicon was digested with EcoRI and BamHI and cloned into the plasmid vector pBC-SK (Stratagene) to give recombinant plasmid pJDD-V2. After confirmatory sequencing, to rule out the presence of unwanted mutations introduced by PCR, the 0.8 kb NdeIBamHI fragment of pJDD-V2, containing the blaVIM-2 gene, was subcloned into the T7-based expression vector pET-9a (Novagen) to obtain plasmid pET-9/VIM-2.
Expression experiments
E. coli BL21(DE3)(pET-9/VIM-2) was grown aerobically at 37°C in 100 mL of BSB medium containing kanamycin, 50 mg/L. When the OD at 600 nm reached a value of 0.8, the culture was split into two and to one subculture isopropyl-ß-D-thiogalactopyranoside (IPTG) (final concentration 1 mM) was added. ß-Lactamase activity was monitored spectrophotometrically, using 100 µM imipenem as substrate in 10 mM HEPES buffer (pH 7.5) (HB), at 30°C, both in cell extracts and in culture supernatants from samples obtained at different times. Cell extracts were prepared by centrifuging the culture, resuspending the cells in the same volume of HB, and disrupting them by sonication [five cycles, 20 s for each cycle, at 45 W, using a B. Braun Labsonic L sonicator (Melsungen, Germany)]. The amount of enzyme was calculated on the basis of the following kinetic parameters for imipenem: kcat = 34/s and Km = 9 µM.
Purification of VIM-2
The VIM-2 enzyme produced by E. coli BL21(DE3)(pET-9/VIM-2) was purified from the culture supernatants of 200 mL stationary phase cultures grown aerobically at 37°C in BSB medium. Cells were removed by centrifugation (10 000g, for 30 min at 4°C) and solid ammonium sulphate was added to the supernatant to achieve a 50% saturation. After 1 h of gentle stirring at 4°C, the sample was centrifuged (13 000g, for 1 h at 4°C). Solid ammonium sulphate was added to the clarified supernatant to 80% saturation, and the precipitate, collected as described above, was solubilized in 20 mM triethanolamine (pH 7.2) (1/20 of the original volume) and loaded (flow rate 2 mL/min) onto an HR column (5 x 1.6 cm) packed with 10 mL of Source 15Q gel (AmershamPharmacia Biotech, Uppsala, Sweden), previously equilibrated with the same buffer. Elution was performed with the same buffer using a linear NaCl gradient (01 M in 200 mL), at a flow rate of 2 mL/min. Fractions containing ß-lactamase activity were pooled and concentrated 10-fold using a Centriplus YM10 system (Millipore, Bedford, MA, USA). The concentrated sample was then injected onto a Superdex 75 HR 10/30 column (AmershamPharmacia Biotech) equilibrated with HB containing 50 µM ZnCl2 and 0.2 M NaCl, and proteins were eluted in the same buffer at a flow rate of 0.8 mL/min. Fractions containing ß-lactamase activity were pooled and stored at 20°C. During the purification procedure the presence of ß-lactamase activity was monitored as described above. Protein concentration in solution was determined with the Bio-Rad Protein assay (Bio-Rad, Richmond, CA, USA), using bovine serum albumin as the standard. The molar extinction coefficient at 280 nm of the purified enzyme was determined as the average of values obtained by the colorimetric protein concentration determination and by theoretical calculation.23
Protein electrophoretic techniques
SDSPAGE was performed according to Laemmli,24 using final acrylamide concentrations of 12% and 5% (w/v) for the separating and the stacking gels, respectively. After electrophoresis the protein bands were stained with Coomassie Brilliant Blue R-250. Analytical isoelectric focusing (IEF) of the purified protein and detection of enzyme activity were performed as described previously.16
Gel-permeation chromatography
Gel-permeation chromatography to determine the molecular mass of the native VIM-2 enzyme was carried out on a Superdex 75 HR 10/30 column (AmershamPharmacia Biotech) equilibrated with HB containing 0.15 M NaCl, to prevent unwanted proteincolumn matrix interactions. The purified enzyme (100 µL, at a concentration of 0.5 mg/mL) was eluted in the same buffer at a flow rate 0.8 mL/min. The low-range gel filtration calibration kit (AmershamPharmacia Biotech) was used for column calibration. Apparent partition coefficients (Kav) were calculated as described previously.25
N terminus sequencing and electrospray mass spectrometry
The amino-terminal sequence of the purified VIM-2 protein was determined using a gas-phase sequencer (Procise-492, Applied Biosystems, Foster City, CA, USA), after redissolving the protein (50 pmol) in 0.1% (v/v) trifluoroacetic acid in water and loading the sample onto a PVDF membrane (Millipore Corp.). Electrospray mass spectrometry was carried out using a PE-Sciex API III triple quadrupole mass spectrometer equipped with an ion-spray source (Perkin-Elmer, Rahway, NJ, USA). The sample (150 pmoles) was redissolved in 1% (v/v) potassium formate/70% (v/v) acetonitrile in water and injected into the source of the mass spectrometer at a flow rate of 20 µL/min. Source and cone voltages were 5.5 kV and 60 V, respectively. The source temperature was kept at 50°C. Twenty-four scans covering 8001800 atomic mass units were accumulated and data were analysed with the software delivered with the instrument.
Determination of kinetic parameters
Substrate hydrolysis by the purified enzymes was monitored by following the absorbance variation, at 30°C, using a Cary 100 UV-Vis spectrophotometer (Varian Instruments, Walnut Creek, CA, USA), in a total reaction volume of 500 µL. The sources, wavelengths, changes in extinction coefficients and reaction buffer (HB containing 50 µM ZnCl2) used in the spectrophotometric assays were the same as described previously.11,18 The final enzyme concentrations ranged from 4.2 to 84 nM. Enzyme dilutions were prepared in the reaction buffer supplemented with 20 mg/L BSA, and were discarded after each working session. The steady-state kinetic parameters (Km and kcat) were determined under initial-rate conditions using the HanesWoolf plot26 and, when standard deviation values exceeded 5%, they were also verified by the analysis of the complete hydrolysis time-courses as described by De Meester et al.27 Km values lower than 10 µM were measured as inhibition constants (Kis) with a competitive model, using 100 µM nitrocefin as the reporter substrate, as described previously.18 Purified VIM-1 enzyme for kinetic measurements was prepared as described previously.18
Inactivation by chelating agents
Inactivation time-courses were monitored by following the hydrolysis of 150 µM imipenem in the presence of different concentrations of EDTA, dipicolinic acid or 1,10-o-phenanthroline. Reactions were carried out at 30°C in HB in a final reaction volume of 500 µL. Enzyme dilutions were prepared in the same buffer supplemented with 20 mg/L BSA, and the final enzyme concentration was 2.1 nM. Pseudo-first-order inactivation rate constants (ki) were determined and the inactivation efficiencies (k+2/K) were calculated according to the proposed model.28
Molecular modelling
VIM-1 and VIM-2 structural models were based on the available X-ray diffraction three-dimensional structure of CcrA from Bacteroides fragilis.5,29 Molecular models were built by knowledge-based modelling using the HOMOLOGY module of the Insight II software (Molecular Simulations, San Diego, CA, USA) running on a Silicon Graphics Indy workstation (Silicon Graphics Inc., Mountain View, CA, USA). Histidines present at the active site were taken as neutral; for the others, the tautomeric form of the imidazole ring was chosen according to the X-ray structure, after geometric analysis of the potential hydrogen bonds. The zinc-coordinated cysteine was in the thiolate form. Other titratable sites were assigned their standard protonation states at pH 7. Hydrogen atoms were added to the structure using the PROTONATE module of the AMBER version 4.1 software.30 Atomic charges of the AMBER version 4.1 all-atom library were generally used in the calculations, except for the residues of the active site. Atomic point charges for the zinc ions, the zinc coordinating residues and active-site water molecules were those proposed by Banci et al.31 Docking experiments were carried out with the Insight II software using the benzylpenicillin structure optimized by AM1 semi-empirical methods. The corresponding complex structures were then optimized by molecular mechanics methods using AMBER version 4.1, first by steepest descent energy minimization of atoms with a force >500 kcal/mol/Å and then by conjugate gradient energy minimization until the rms gradient was <0.1 kcal/mol. Graphics were realized with MOLMOL molecular graphics software.32 The BBL numbering scheme is used throughout this paper.33
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Results and discussion |
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Under the experimental conditions employed, VIM-2 hydrolysed all the tested compounds except aztreonam. The individual kinetic parameters (Km and kcat) of VIM-2 with several substrates, and a comparison with those of VIM-1, are reported in Table 2.
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Hydrolytic efficiencies of VIM-2 with cephalosporins were highly variable, with values of kcat/Km ratios ranging from 5 x 104 to 1 x 107/M·s. Except for cefepime, the Km values were quite low and usually lower than those of penicillins, whereas the turnover rates exhibited an overall higher variability, which often directly influenced the hydrolytic efficiencies. With cefepime, only a pseudo-first-order rate constant was measurable, since the initial velocities remained proportional to substrate concentration up to a value of 400 µM. Compared with VIM-1, both the kcat and Km values of VIM-2 tended to be lower (with some exceptions for cefepime, nitrocefin and moxalactam). With some substrates (e.g. cefaloridine, cefalothin, ceftazidime and cefpirome) the differences in individual kinetic parameters again balanced out, resulting in similar hydrolytic efficiencies for the two enzymes, whereas with other substrates (e.g. cefoxitin, cefuroxime, cefotaxime and moxalactam) the kcat/Km ratios were also different. The largest difference in hydrolytic efficiencies (almost 40-fold higher for VIM-1) was observed with cefepime, a finding that was consistent with in vitro susceptibility data.16,17
VIM-2 exhibited high hydrolytic efficiencies with all carbapenems (kcat/Km ratios were in the range 2.55.5 x 106/M·s), resulting from a combination of very low Km values and relatively low turnover rates. Overall this behaviour was similar to that observed for VIM-1, and differentiates the VIM-type enzymes from all other zinc-ß-lactamases that achieve similar hydrolytic efficiencies toward carbapenems as a result of higher turnover rates combined with higher Km values.18 Notwithstanding this common trend, differences in individual kinetic constants between VIM-2 and VIM-1 were also apparent with carbapenems, resulting in different hydrolytic efficiencies. In particular, imipenem and meropenem were hydrolysed more efficiently by VIM-2 (30- and 10-fold, respectively), due to a higher turnover rate and to a lower Km value, respectively.
Serine-ß-lactamase inactivators were hydrolysed by VIM-2, with individual kinetic parameters and hydrolytic efficiencies that are only slightly different from those of VIM-1.
The present data are very similar to those previously reported for VIM-217 for some substrates (e.g. cefuroxime), while being significantly different for others (e.g. most cephalosporins). In particular, the kcat values measured in this work tended to be higher than those previously reported, except for cefuroxime and ceftazidime (for the latter substrate the kcat was actually much lower), whereas smaller differences were observed between Km values.17 These discrepancies are likely to reflect differences in the experimental conditions (different buffer systems, HEPES versus sodium cacodylate, at pH 7.5 versus 6.5, respectively, were used) and, possibly, a different degree of purity or of specific activity of the enzyme preparation. They also emphasize the importance of carrying out comparative kinetic studies under identical experimental conditions.
VIM-2 was inactivated by EDTA, o-phenanthroline and dipicolinic acid. The inactivation time-courses followed pseudo-first-order kinetics, and the inactivation rates varied proportionally with the chelating agent concentrations within the experimental range, allowing only the measurement of the k+2/K ratio (EDTA: 2.5/M·s; o-phenanthroline: 860/M·s; dipicolinic acid: 460/M·s), representing the inactivation efficiency. Although the mode of interaction appeared to follow a mechanism similar to that observed with other metallo-ß-lactamases of subclass B1,11,18,35 VIM-2 was more efficiently inactivated than VIM-118 by all three chelators (the k+2/K ratios were 4- to 20-fold higher for o-phenanthroline and dipicolinic acid, respectively), indicating that zinc ions are probably more tightly bound in the latter enzyme.
At the sequence level VIM-1 and VIM-2 are 93% identical and can be aligned without the introduction of major gaps with all the subclass B1 ß-lactamases,33 including those (Bc-II, CcrA and IMP-1) for which three-dimensional structures are available.4,5,7,29 Of the latter enzymes, CcrA was chosen as the basis for construction of VIM-1 and VIM-2 structural models, since although Bc-II exhibits a higher similarity, the CcrA structure has better defined coordinates for the loop between strands 3 and 4.29 Compared with CcrA, the differences in terms of deletions/insertions for VIM-1 and VIM-2 consist of: (i) the lack of one residue (W64) in the L1 loop between strands 3 and 4; and (ii) the insertion of two residues (L172 and E173) in the L2 loop, between helix 3 and strand 8 (Figure 3).
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Comparative biochemical analysis carried out under the same experimental conditions allowed us to confirm that there are notable differences between VIM-1 and VIM-2 in their interactions with several ß-lactam substrates and also with metal chelators. This means that at least some of the amino acid differences between the two proteins must have functional significance. For this reason, the two natural VIM variants present a relevant model for understanding the roles of specific residues in the mechanism of zinc-ß-lactamases. It should be noted that although the two /ß domains constituting the protein are similar in size, most of the differences between the two enzymes (12 of 17) are clustered in the second domain, whereas the first domain is more strongly conserved (it contains only five differences, of which three are located at the N terminus and are not likely to be relevant to the protein fold). Since VIM-1 and VIM-2 are probably derived from a common ancestor, the above observation suggests that the C-terminal domain could evolve at a faster pace to give different specificities to this type of enzyme.
Molecular modelling suggests a possible role for some of the different residues and, on the basis of these indications, we are currently generating specific mutants to investigate this point. Resolution of the three-dimensional structure of the VIM enzymes is also underway, and will provide an essential contribution to the understanding of the mechanistic properties of these clinically relevant enzymes. The functional diversity encountered among metallo-ß-lactamases produced by opportunistic pathogens could be critical to the development of effective broad-spectrum inactivators of these enzymes.
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Acknowledgements |
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Footnotes |
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