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J. Biol. Chem., Vol. 277, Issue 27, 24744-24752, July 5, 2002

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Characterization of Monomeric L1 Metallo--lactamase and the Role of the N-terminal Extension in Negative Cooperativity and Antibiotic Hydrolysis^*

Alan M. Simm^§, Catherine S. Higgins, Anne L. Carenbauer^¶, Michael W. Crowder^¶, John H. Bateson, Peter M. Bennett, Anthony R. Clarke^**, Stephen E. Halford^**, and Timothy R. Walsh

From the  Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom, the ^¶ Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, the  Department of Medicinal Chemistry, GlaxoSmithKline Pharmaceuticals, Harlow, Essex CM19 5AW, United Kingdom, and the ^** Department of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom

Received for publication, February 14, 2002, and in revised form, April 8, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REACTION SCHEME 1 REFERENCES

The L1 metallo--lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in -lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 -lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form but demonstrated greatly reduced rates of -lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higher K_m values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K_m values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for -lactam hydrolysis, it is clearly important to its activity and substrate specificity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REACTION SCHEME 1 REFERENCES

Stenotrophomonas maltophilia is an opportunistic pathogen that is emerging as a significant cause of nosocomial infections, particularly among immunocompromised patients (1, 2). S. maltophilia is inherently resistant to most antibacterial drugs, including most, if not all, -lactams (3, 4). -Lactam resistance in this organism is mainly mediated by the inducible expression of two -lactamases (designated L1 and L2) (5, 6). These are bacterial enzymes that hydrolyze -lactam antibiotics, rendering them ineffective as inhibitors of bacterial cell wall synthesis (7). -Lactamases, of which more than 400 have been identified, have been classified according to substrate specificity, inhibitor profile, and requirement of a metal cofactor at the active site (8). The metallo--lactamases (Group 3) are distinct from the majority of -lactamases in that they require zinc at the active site for maximal activity. They hydrolyze carbapenems such as imipenem and meropenem, which, apart from one or two exceptions, are poorly hydrolyzed by serine -lactamases (8, 9). Metallo--lactamases are also insensitive to many serine -lactamase inhibitors, such as clavulanic acid and sulbactam (8). To date, there are no clinically useful inhibitors of metallo--lactamases (9, 10).

Chromosomal genes encoding metallo--lactamases have been sequenced from a number of organisms including Aeromonas spp. (11, 12), Bacillus cereus (13), Bacteroides fragilis (14), Chryseobacterium spp. (15, 16), Legionella gormanii (17), S. maltophilia (18), and Caulobacter crescentus (19). The mobile metallo--lactamase genes encoding for IMP-1 and VIM-1 (and variants thereof) have been disseminated by both plasmid- and integron-mediated systems and have been identified in a number of organisms, including Pseudomonas aeruginosa (20, 21), Acinetobacter baumannii (22), and Serratia marscescens (23). Crystal structures have been determined for four metallo--lactamases: B. cereus (BcII) (24-26), B. fragilis (CcrA) (27, 28), P. aeruginosa (IMP-1) (29), and S. maltophilia (L1) (30). These structures all contain an / fold, generally contain the zinc-binding motif His-X-His-X-Asp, and all bind either one or two atoms of zinc at the active site.

L1 is distinct from all other known metallo--lactamases in the following respects. (i) The functional enzyme is a tetramer (5, 30, 31). (ii) Its monomer is 4-5 kDa larger than the others. (iii) It contains a number of substitutions at sites thought to be important for metallo--lactamase function and particularly in zinc-binding ligands (30). From the crystal structure of L1 (30) it is known that the tetramer structure contains three sets of intersubunit interactions. The principal contact between the A and B subunits is a hydrophobic interaction between the side-chain of Met-140 from chain A and a pocket formed by Leu-122, Pro-162, Tyr-199, and Pro-200 from chain B. An identical interaction binds the C and D subunits. The A and C chains (as well as B and D chains) interact in two regions, most importantly between the extended N-terminal residues of each chain where leucine residues 5 and 8 dock into apolar cavities formed by Ala-10, Tyr-11 and Ala-15, and Met-56 to His-62. This extension of the N-terminal region of ~20 amino acids is unique to L1 (24), not BceII (25, 26), CcrA (27, 28), or IMP-1 (29). The second, more limited, interaction is between the side chain of Pro-132A with residues Pro-132C and Arg-116C. It has been proposed that interactions involving residues Met-140 and the hydrophobic N-terminal residues Leu-5, Leu-8, and Tyr-11 were important in maintaining tetramer stability (30). The monomeric structure of L1 is shown in Figs. 1 and 2, and these residues are labeled. Docking studies, in which the substrates imipenem, ceftazidime, and ampicillin were docked into the active site of L1 and subjected to energy minimization, suggested interactions between the residues Trp-17 and Tyr-11 with the pyridine ring of ceftazidime and the aromatic substituent of ampicillin, respectively (30). In addition, the positioning of Trp-17 near the active site led to the suggestion that this residue makes stabilizing edge-face contacts with the Zn(II) ligand, His-225 (30).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   The crystal structure of the monomer subunit of the L1 metallo--lactamase from S. maltophilia (PDB, 1sml) showing the N-terminal extension and the positions of the residues of interest. -Helices are in red and -sheets in yellow. The structure was rendered using the InsightII package (Molecular Simulations, Inc.).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Interactions between subunits of the L1 tetrameric structure involving residues in the extended N-terminal region. The -carbon trace of the tetrameric protein is shown with chains A and C in yellow and blue, respectively (chains B and D are in white). Amino acid residues 2-20 in the N-terminal regions are highlighted and enlarged. Van der Waals surfaces of tyrosine 11 and leucines 5 and 8 are shown for chains A and C. The aromatic ring surface of tyrosine 11 fits in the hydrophobic pocket between the two leucine residues. The structure was rendered using the InsightII package (Molecular Simulations Inc.).

The object of this study was to examine the predictions made about the role of Met-140 and the N terminus by investigating the properties of a M140D mutant enzyme, an N-terminal deletion (N-Del)¹ mutant lacking the first 20 amino acids of the mature protein, and a set of discrete site-directed mutants at the N terminus; residues Leu-5, Leu-8, Tyr-11, and Trp-17. Understanding the roles of specific residues in L1, particularly in terms of substrate binding, is expected to contribute significantly to the rational design of therapeutic inhibitors of the L1 metallo--lactamase. We report the first study on L1 that probes the roles of specific residues and the first production of an L1 monomeric enzyme. This study also identifies negatively cooperative kinetics in the tetrameric enzyme and demonstrates that this effect is mediated through N-terminal subunit interactions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REACTION SCHEME 1 REFERENCES

Unless otherwise stated, media used were either nutrient broth or nutrient agar (Oxoid plc., Basingstoke, UK). PCR primers were purchased from Sigma-Genosys Ltd. and used without modification. -Lactams used were nitrocefin (BD PharMingen), imipenem (Merck Sharpe and Dohme, Huddesdon, UK), ceftazidime (Glaxo Laboratories Ltd., Greenford, UK), meropenem (Zeneca Pharmaceuticals, Macclesfield, UK), penicillin G, cefoxitin, cephaloridine, cefmetazole, cefotaxime, and ampicillin (all from Sigma). All other general reagents were from Sigma or from BDH, both of Poole, UK.

Generation of Site-directed Mutants-- The L1 gene (L1-WT), originally cloned from S. maltophilia strain IID 1275 on plasmid pUB5811 (18), was amplified by PCR using "L1-F" and "L1-R" primers (Table I). PCR was performed as described previously (32). These primers were designed to target both ends of the published L1 sequence (18) to enable amplification of the full gene. A deletion mutant of L1, lacking the N-terminal 20 amino acids of the mature protein (i.e. in addition to the cleaved signal peptide) was made by PCR using the "NDel-F" and "L1-R" primers. Specific site-directed mutants of L1 were created by PCR, using the method of Ho et al. (33), with pUB5811 as a template (18). Internal mutagenic primers (Table I), containing the desired mutation, were used in conjunction with the L1-F and L1-R primers to create two part-length amplicons. These two amplicons were mixed, denatured, and annealed, and the products were used as templates for a subsequent PCR reaction using the L1-F and L1-R primers to generate the required full-length L1 mutant.

PCR products were purified using a QIAquick PCR purification kit (Qiagen Ltd., Crawley, UK) and sequenced on both strands with an ABI Prism 377 automatic sequencer (PerkinElmer, Warrington, UK) using dye termination chemistry according to the manufacturer's instructions to confirm the insertion of the mutation. DNA and protein sequences were compared using the Lasergene suite of programs (DNASTAR Inc, Madison, WI). DNA sequencing of the L1 mutants L5A/L8A and W17S confirmed that only the desired mutations were present; in the case of the N-Del mutant, no mutations in the remaining sequence were found.

Despite many attempts, a single Y11S mutant could not be recovered. Further attempts to mutate Tyr-11 to threonine or phenylalanine were also unsuccessful. Analysis of numerous transformants from these cloning reactions generally revealed one of the three following outcomes. (i) The mutation was unsuccessful, and the DNA sequence recovered was wild-type. (ii) A Tyr-11 mutation had been introduced, but the gene had inserted in the wrong orientation in the vector for expression. Attempts to switch the orientation were unsuccessful. (iii) A Tyr-11 mutation was introduced, and the mutant gene was inserted in the correct orientation in the vector, but the desired mutation was accompanied by at least one additional mutation. Examples of the mutants recovered are: Y11S + A15T, Y11S + T212A, Y11S + N134T, Y11S + A69V + I138N, Y11T + V13E, Y11T + S16T, and Y11F + R9G. In this study we were interested in examining the involvement of the N-terminal region of L1. For this reason, a Tyr-11 mutation was chosen for study, which contained a second mutation in this region, namely Y11S + A15T.

Given the ease with which all but the Tyr-11 substitutions were created, failure to generate mutations at the Tyr-11 locus in isolation is surprising. It seems likely that a mutation at this locus somehow creates a protein that is lethal to the cell; in which case, the mutations that are recovered in addition to the Tyr-11 mutation must suppress the lethal tendency. We have no sensible hypothesis for how this might be achieved. Resolution of this question clearly requires detailed study of L1 folding.

Cloning, Overexpression, and Purification-- PCR amplicons were TA-cloned into the pTrcHis2-TOPO vector (Invitrogen), and recombinant molecules were transformed into Escherichia coli TOP10 One Shot competent cells (Invitrogen), following the manufacturer's guidelines. The presence of recombinant L1 clones was confirmed by PCR. The L1 mutant proteins were overexpressed and purified by fast protein liquid chromatography (FPLC), as described previously (34). The gel filtration column was calibrated using immunoglobulin G (BioRad Laboratories) (molecular mass, 150,000 Da; elution volume, 260 ml), bovine serum albumin (Sigma) (molecular mass, 67,000 Da; elution volume, 305 ml), and carbonic anhydrase (Sigma) (molecular mass, 29,000 Da; elution volume, 340 ml).

Elution of L1 from the gel filtration column was followed with an antibody to the native L1 protein using a standard ELISA method (35). The L1 antibody was raised by injecting New Zealand White rabbits with a 2 mg/ml solution of pure tetrameric L1 in phosphate-buffered saline at two weekly intervals over a period of 8 weeks. Animals were anesthetized and bled via cardiac puncture. (This procedure was performed by Affiniti Research Products Ltd.) Serum was separated and assayed using the above ELISA technique. The maximum working dilution of this polyclonal serum in ELISA assays was 1:10⁶.

Peak fractions containing L1 protein were pooled and used for further analysis. Electrospray mass spectrometry (using a VG Quattro Mass Spectrometer) was used to assess molecular weight and protein purity. N-terminal sequencing was performed with an Applied Biosystems 477A amino acid analyser. Protein for each technique was prepared as described by Winston and Fitzgerald (36), starting with 500 µl of 5 µM enzyme.

Preparation of the M140D Mutant-- The overexpression plasmid for L1, pUB5832, was digested with NdeI and HindIII, and the resulting ~900-bp piece was gel-purified and ligated using T4 ligase into pUC19, which was also digested with NdeI and HindIII, to yield the cloning plasmid pL1PUC19. Mutations were introduced into the L1 gene by using the overlap extension method of Ho et al. (33). The oligonucleotides used for the preparation of the mutants are shown in Table I. pUCMSZfor and M13Rev were used as the universal forward and reverse primers, respectively. The ~900-bp PCR products were digested with NdeI and HindIII and ligated into pUC19. The DNA sequences were analyzed by the Biosynthesis and Sequencing Facility in the Dept. of Biological Chemistry at Johns Hopkins University. After confirmation of the sequence, the mutated pL1PUC19 plasmid was digested with NdeI and HindIII, and the 900 bp, mutated L1 gene was gel-purified and ligated into pET26b to create the mutant overexpression plasmids. E. coli BL21(DE3)pLysS cells were transformed with the mutated overexpression plasmids, and large scale (4 liter) preparations of the L1 M140D mutant enzyme were performed as described previously (34). Protein purity was assessed by SDS-PAGE.

Steady State Kinetics-- -Lactamase assays and steady state kinetics were carried out as described previously (34). Extinction coefficients and wavelengths for the -lactams used were: nitrocefin, 17,400 AU M¹ cm¹ (at 482 nm); imipenem, 7,000 AU M¹ cm¹ (at 299 nm); ceftazidime, 9,000 AU M¹ cm¹ (at 265 nm); and ampicillin, 809 AU M¹ cm¹ (at 233 nm). Antibiotic solutions were prepared in 50 mM sodium cacodylate, pH 7.0, containing 100 µM ZnCl₂. Enzyme concentrations were determined using extinction coefficients (at 280 nm), which were calculated from the amino acid sequence of each mature L1 enzyme using the Gill and von Hippel algorithm (37) as follows: L1WT, 38,930 AU M¹ cm¹; N-Del, 31,960 AU M¹ cm¹; W17S, 33,240 AU M¹ cm¹; L5A/L8A, 38,930 AU M¹ cm¹; Y11S (+A15T), 37,650 AU M¹ cm¹.

Zinc Content-- Purified L1 was dialyzed against 500 volumes of 50 mM cacodylate, pH 6.0 (24 h, 20 °C), with one change of dialysis buffer. The enzyme was concentrated to 10-30 µM in 2.5 ml using a Centricon Plus10 filter unit (Millipore). Concentrated enzyme was mixed with an equal volume of 11.4 M nitric acid (BDH, Aristar Grade) and incubated for 18 h, 20 °C to hydrolyze the protein. The zinc content of this mixture (as ppm) was assayed in a Unicam 919 atomic absorption spectrometer at 213.9 nm, and the data were reported as zinc ions per subunit.

Ultracentrifugation-- Samples (110 µl) of L1 wild-type and mutant enzymes were examined by centrifugation to equilibrium at 20 °C in 3-mm columns in a Beckman XLA analytical ultracentrifuge. After centrifugation for the requisite times, typically 16 and 20 h at 10,000 rpm followed by the same time intervals at 15,000 rpm, the radial distribution of the protein was measured by absorption at 280 nm. The distributions recorded 4-h apart duplicated each other, thus confirming that the sedimentation was at equilibrium. Another absorption record was taken after extending the centrifugation for 6 h at 40,000 rpm. The absorption at the meniscus in the latter record was used as a fixed offset in the subsequent analysis with the ORIGIN software from Beckman (38). The analysis, fitted by non-linear regression for either single or multiple absorption records to obtain the best fit for M (molecular mass), is shown in Equation 1,

(Eq. 1)

where A_r and A₀ are the absorbances at radius r and at the reference radius r₀; , angular velocity; R, gas constant; T, temperature; , partial specific volume (0.734 ml/g for L1); , buffer density (1.008 g/ml for the buffer used here); and B, baseline offset (determined as above). Partial specific volumes and buffer densities were calculated with SENDTERP (Ref. 39, alpha.bbri.org/rasmb/spin/ms_dos/sednterp-philo/).

Modeling of the N-terminal Extension in the Monomer Enzyme-- The possible conformations available to the unrestrained N-terminal extension of L1 (as would be likely to exist in the monomer) were computed using the ab-initio evolutionary Monte Carlo technique of Gibbs et al. (40). For the first 19 N-terminal amino acids, an initial set of structures was generated with backbone - geometries applied randomly. Each structure was then subjected to random mutations of its backbone - angles to form 10,000 daughter molecules. After energy minimization the most stable 1,000 structures are retained as the next generation of parent structures. This process was repeated for 7 generations where the 1,000 lowest energy structures are retained as the best solution to the conformational search.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REACTION SCHEME 1 REFERENCES

Purification and Quaternary Structure of L1 Mutant Enzymes-- For each mutant a protein was purified that consisted of a single band on an SDS-PAGE gel. On the basis of size exclusion chromatography the calculated native molecular masses of the parent enzyme, L1, and mutant proteins were as follows: wild-type L1 (117,000 Da), L5A/L8A mutant (108,000 Da), Y11S (+A15T) mutant (108,000 Da), W17S mutant (117,000 Da), N-deletion mutant (96,000 Da), and M140D mutant (28,000). The purity and monomeric molecular weights of wild-type and all mutant enzymes were also determined by mass spectrometry. For all preparations protein purity was >95%, and all molecular masses were consistent with values predicted from the primary sequences. N-terminal sequencing of the wild-type L1 enzyme and the N-deletion mutant gave sequences of AEVPLPQ and MAPLQIA, respectively (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Amino acid sequence of the mature wild-type and mutant proteins (i.e. after cleavage of the signal peptide). The N-Del mutant lacks the first 20 amino acids of the mature L1 protein.

The sedimentation equilibrium data for the N-deletion, and M140D enzymes are shown in Fig. 4a and b, respectively. Data for L1-wild type was reported in Ref. 30, and the curves for L5A/L8A and Y11S (+A15T) are essentially the same as for the wild-type enzyme. The molecular weights were as follows: N-deletion mutant enzyme, 105,503 (S.E. 97,836-112,967); L5A/L8A enzyme, 110,058 (S.E. 106,482-114,000); Y11S (+A15T), 108,480 (S.E. 103,874-112,987); M140D, 27,811 (S.E. 26,983-28,648). All the fitted molecular weights are consistent with tetrameric proteins with the exception of M140D, which is clearly monomeric. Attempts to fit the M140D data to mixed equilibria models in which the monomer was in equilibrium with tetramer or dimer species were not successful.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Sedimentation equilibrium. Samples of 0.35 mg/ml N-Del mutant LI enzyme (a) and M140D enzyme (b), in 50 mM cacodylate buffer pH 7.0, were sedimented for 20 h at 20 °C at either 10,000 (a) or 15,000 rpm (b), respectively. In both a and b, the data points in the main panels denote the absorbance at 280 nm as a function of the centrifugal radius, and the solid line denotes the best fit to M in Equation 1. The best fit to the data from the N-Del mutant (a) was with M = 105,503 Da and that to the data from the M140D mutant (b) was with M = 27,811 Da. The upper panels in both a and b denote the residuals between the data and the best fits.

Zinc Content-- The zinc contents of wild-type L1 and each mutant enzyme were determined. Wild-type L1 binds 1.8 ± 0.1 zinc ions per subunit, in agreement with values published by Crowder et al. (41). The L5A/L8A, Y11S (+A15T), and M140D mutant enzymes bind 1.9 ± 0.1, 1.9 ± 0.2, and 1.9 ± 0.1 zinc ions per subunit, respectively, indicating that in all these enzyme variants there is full metal occupancy in both binding sites. The W17S and N-deletion mutant enzymes bind 1.6 ± 0.2 and 0.8 ± 0.2 zinc ions per subunit, respectively.

Steady State Kinetics of Wild-type L1 and L1 Mutant Enzymes-- Steady state kinetic analyses were performed on multiple preparations of the wild-type L1 and each mutant enzyme, and the results for hydrolysis of nitrocefin, imipenem, ceftazidime, and ampicillin are shown in Table II. The parent enzyme, L1, exhibited comparable kinetic values for imipenem, nitrocefin, and ampicillin compared with those published previously (34, 41). The kinetic values for ceftazidime for the wild-type enzyme have not been published previously.

N-Del displays markedly different kinetics compared with those of wild-type enzyme and hydrolyzes most tested -lactams significantly more slowly. The K_m of N-Del for imipenem is similar to that of wild-type, whereas the k_cat is decreased by 14-fold. In contrast, the K_m for nitrocefin is increased significantly (20-fold), whereas the k_cat remains unchanged. The specificity constants of the N-Del mutant enzyme for both nitrocefin and imipenem are much lower than those for the parent enzyme. Although the N-Del mutant enzyme hydrolyzes ampicillin, the rates obtained were too low to obtain accurate kinetic parameters, and the N-Del mutant enzyme does not hydrolyze ceftazidime at detectable rates.

The kinetic parameters of the mutant enzymes containing amino acid substitutions are different from those of the wild-type enzyme. With ampicillin as a substrate, the K_m values are increased in all cases (3-5-fold), with the Y11S (+A15T) mutant showing the largest increase in K_m. The k_cat values of the same enzymes decrease compared with wild-type enzyme (3-5-fold) (Table II). When ceftazidime is the substrate, the K_m values of the mutant enzymes are generally very similar to that of wild-type enzyme, while the k_cat values of all mutant enzymes are greatly decreased (27-42-fold). The K_m values for imipenem are generally comparable with wild-type enzyme or slightly increased. The k_cat values are also comparable or slightly reduced with wild-type. When nitrocefin is the substrate, the K_m values are increased in all cases (10-17-fold), as are the k_cat values (5-8-fold). The specificity constants (k_cat/K_m) for imipenem and nitrocefin for the mutant enzymes are slightly reduced compared with that of the wild-type enzyme, whereas the values for ampicillin, and particularly ceftazidime, are much lower.

Kinetics of Cooperativity of Native L1 Enzyme, the N-terminal Deletion Mutant and Monomer L1-- Using nitrocefin as the reporter substrate, the results of steady state rate determinations at different substrate concentrations were transformed into a Hill plot (log(v/V_max  v) versus log[S]) (42) for the wild-type L1 enzyme, the N-terminal deletion enzyme, and the monomeric L1 enzyme. This plot is shown in Fig. 5. The wild-type enzyme was negatively cooperative with a Hill coefficient of 0.71. Neither the monomer nor the tetrameric N-deletion enzyme showed cooperativity (Hill coefficients of 1.00 and 0.98 for N-deletion and M140D mutants, respectively).

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Hill plot of steady state kinetic data for L1 wild-type enzyme, N-Del, and M140D mutants. Data are for nitrocefin hydrolysis.

Modeling of the N-terminal Extension in Monomer L1-- After energy minimization of the N-terminal conformers generated from the monomer structure, the potential energies of the 100 most stable conformers were investigated. Fig. 6 shows the frequency distribution of these energies. There were a wide variety of possible conformers within a very narrow energy range suggesting that the N-terminal extension is highly mobile when unconstrained by interchain interactions. There are 25 conformers all with energies between 105.367 kJ/mol (the most stable conformer) and 100.9426 kJ/mol. None of these conformers were extended forms as seen in the wild-type enzyme. All were variants of a folded loop. The structure of the most stable conformer is shown in Fig. 7 and is typical of the sort of conformation adopted by all 100 most stable geometries.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Frequency distribution of the 100 most stable N-terminal conformers generated by the Monte Carlo folding algorithm of Gibbs et al. (40). The figure on the x-axis represents the lower value of the energy range, and the number associated with each column is the number of conformers having an energy between that value and the lowest range of the next class.

View larger version (74K): [in this window] [in a new window]   Fig. 7.   The most stable monomer N-terminal conformer generated by the algorithm of Gibbs et al. (40) (white) compared with the L1 crystal structure (red; PDB, 1sml). Each fragment comprised the first 20 N-terminal amino acids, and the atoms of residue 20 have been superimposed. The two zinc atoms from the native L1 structure have been included to indicate the position of the active site.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REACTION SCHEME 1 REFERENCES

The crystal structures of four metallo--lactamases have been determined (24-30), which when supplemented by pre-steady state kinetic analyses have enabled reaction mechanisms for the hydrolysis of -lactams to be predicted for some enzymes (43-46). In the case of the metallo--lactamases CcrA from B. fragilis and BcII from B. cereus, the predicted mechanisms have also been examined by the use of site-directed mutants (47-50). It is clear from all of these studies that the mechanism of -lactam hydrolysis and the residues involved differs from one enzyme to another. Accordingly, it is important to investigate how each enzyme binds and hydrolyzes substrates to understand the class as a whole. This report presents the first study of the L1 metallo--lactamase that investigates the roles that specific residues play in its catalytic activity.

Stability of the L1 Tetramer Structure-- L1 is unique among -lactamases in that the functional enzyme is a tetramer (5, 30, 31). A number of interactions are predicted to be involved in the stability of the tetramer structure, including those involving amino acids at the N terminus (30). Considering these predictions, the Y11S (+A15T) mutant might be expected to destabilize these interactions, as might the double mutant L5A/L8A, thus disrupting the tetramer structure. However, in each case, the major protein recovered from gel filtration was the tetrameric enzyme (confirmed by ultracentrifugation), demonstrating that the interactions between subunits involving residues in the first 20 amino acids are not essential for tetramer stability. Ullah et al. (30) proposed that the main interchain interaction stabilizing the tetramer structure involves the side chain of Met-140 on chain A penetrating a hydrophobic pocket on chain B (repeated between chains C/D); other minor interactions were also postulated (30). It is evident from the present study that these interactions are sufficient to maintain integrity of the quaternary structure in solution. Furthermore, disruption of this interaction by the single mutation M140D is sufficient to produce the monomeric enzyme.

Zinc Binding-- The L1 protein normally binds two zinc ions per monomer (41). Although none of the N-terminal residues are predicted to be direct zinc ligands, it has been suggested that Trp-17 can make an edge-face interaction with His-225, a Zn(II) ligand (30). Such an interaction is expected to stabilize the outer coordination sphere of Zn(II). None of the single or double point mutants, and in particular W17S, showed a significant change in zinc content. Hence, we can conclude that any stabilizing effect from Trp-17 is not essential for Zn(II) coordination. As M140D binds 2 zinc ions we can conclude that intersubunit interactions via the N-terminal region are not required for zinc binding. However, the N-Del mutant enzyme binds only one zinc ion per subunit. This could be interpreted to indicate that the loss of the N-terminal 20 amino acids alters the overall structure of L1 to the extent that zinc coordination is adversely affected. However the presence of -lactamase activity in this mutant is strong evidence that the overall protein fold is still intact. There is at present no evidence to indicate which zinc ion is missing in N-Del.

Kinetic Parameters-- A series of docking simulations using imipenem, ampicillin, and ceftazidime were reported with the L1 crystal structure (30). These simulations suggested a series of key interactions between the antibiotic molecule and the enzyme, disruption of which might be expected to influence the kinetic parameters of the enzyme. Interactions involving the N-terminal region were predicted between the aromatic substituent of ampicillin with Tyr-11 and the pyridine ring present in the C3 substituent of ceftazidime with Trp-17 (41). The kinetic parameters reported in Table II for the amino acid-substituted mutant enzymes are broadly consistent with this model. There is a large decrease in the ability of all of these mutant enzymes to hydrolyze ceftazidime. This finding suggests that N terminus residues are involved in ceftazidime catalysis. However, because the detailed molecular mechanism of hydrolysis of ceftazidime by L1 has not as yet been elucidated, any correlation of structure with such effects remains speculative.

The most remarkable difference in kinetic parameters was seen with the monomer enzyme, which exhibited very high K_m values for all antibiotics tested. With the exception of nitrocefin hydrolysis, which we consider is a special case and is discussed below, the monomer k_cat values were significantly reduced implying that the monomer is much less efficient at -lactam turnover. The results of modeling an N-terminal peptide in the absence of interchain stabilizing effects (i.e. the monomer) suggest that the extension is highly mobile and capable of adopting a large number of conformations. Given that the results with single mutations implicate this extension in the normal hydrolysis of substrate, it is probable that substrate binding and possible intermediate stabilization is greatly impaired.

Cooperativity-- The tetrameric wild-type enzyme is clearly negatively cooperative. That this effect is absent in the monomer enzyme is unsurprising. However the effect is also absent from the N-deletion mutant enzyme, which implies that the cooperative effect is mediated through the N-terminal. It is possible that a conformational change in one N-terminal extension induces a change in the interacting chain thereby reducing the substrate binding affinity of the second subunit. The evolutionary advantage of this situation is not clear. Because in a negatively cooperative enzyme, rates at substrate concentrations below the K_m are higher than would be expected in a non-cooperative situation it may be that there is an evolutionary advantage in an enzyme that hydrolyzes low levels of -lactam more efficiently. However it is not certain that -lactams are the natural substrates of these enzymes (51), and in the native situation this cooperativity may have a quite different although as yet unknown role.

Nitrocefin Hydrolysis-- In general estimates of K_m do not give information about the affinity of substrate binding because the relative contribution of different elementary rate constants is not known. However the hydrolysis of nitrocefin by L1 has been the subject of two pre-steady state studies using both colorimetric (45) and fluorescent (46) optical signals. These studies have established that the minimal kinetic mechanism for nitrocefin hydrolysis can be represented as Reaction Scheme 1.

	REACTION SCHEME 1

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REACTION SCHEME 1 REFERENCES

For this reaction mechanism K_m and k_cat are described by the following Equations 2 and 3.

(Eq. 2)

(Eq. 3)

Spencer et al. (46) estimated k₃ as 785 s¹ and k₅ as 11.3 s¹. Under these conditions, where k₂ k₃, Equations 4 and 5 apply.

(Eq. 4)

(Eq. 5)

As the constants k₁, and k₂ in Equation 5 all represent functions of the stability of the ES complex it is possible to infer more about the effects of some of these mutations on the stability of the nitrocefin/L1 complex. In Table II, The K_m values for nitrocefin for the N-Del mutant are ~20-fold higher than for wild-type L1 whereas the k_cat is relatively unchanged. This implies that the ES complex is considerably less stable in this mutant. Similar reasoning can be applied to the M140D and Y11S (A15T) mutations where the increase in k_cat is not sufficient in itself to account for the observed rise in K_m. However, this cannot be taken to imply that it is substrate binding itself that is weaker because without pre-steady state measurements we do not know which of the constants in Equation 5 contribute to the increased K_m. For nitrocefin, there is no evidence that the mutations L5A/L8A and W17S affect the stabilty of ES. This is not necessarily true for other antibiotics as Spencer et al. (46) have shown that different antibiotics can have different hydrolysis mechanisms. The above discussion clearly depends upon the assumption that mutant L1 enzymes hydrolyze -lactams by the same mechanism as the wild-type enzyme. Whereas there is no reason to believe that this is not so for these N-terminal mutants this remains to be demonstrated.

During hydrolysis of nitrocefin by both metallo--lactamases, an enzyme-bound intermediate is formed as the initial product of -lactam ring cleavage. This contains a negative charge on the dihydrothiazine nitrogen atom, originally present in the cephem -lactam ring system (44, 45, 52). It is proposed that this negative charge is stabilized by the large conjugated ring substituent at the C3 position (52) and electrostatic interactions with the positively charged Zn(II) center (30, 45, 52). It has been shown that for nitrocefin k_cat is equivalent to the rate of decay of this stabilized intermediate (46) (see Equation 4).

The increases in the k_cat values for nitrocefin hydrolysis in the L1 mutant enzymes containing amino acid substitutions indicate an increase either in the rate of formation of intermediate and/or in the rate of decay of the intermediate. This could, in principle, stem from the destabilization of the intermediate structure, leading to a more rapid protonation of the anionic nitrogen and hence, product formation. This explanation would suggest that the N terminus of L1 enhances the stability of the nitrocefin intermediate structure and that this contribution is lost upon mutation of N-terminal residues.

This study was undertaken to test the predictions made by Ullah et al. (30) regarding the roles of amino acid residues at the N terminus of the L1 metallo--lactamase. Our findings are consistent with some but not all of these predictions and can be summarized in the following statements. (i) Amino acid residues at the N terminus influence the substrate profile of the enzyme. (ii) The N terminus modifies enzyme activity but is not essential for it. (iii) The N-terminal domain as a whole is important to zinc binding, although single amino acid substitutions have little effect on zinc binding. (iv) Interactions between amino acid residues at the N termini of adjacent subunits are not essential for tetramer formation. (v) The Met-140 residue is solely responsible for tetramer formation, and mutation of this residue produces monomeric enzyme. (vi) Negative cooperative effects seen in wild-type L1 are mediated through the N terminus. The data clearly show the importance of the N terminus of L1 to the enzyme. It clearly assists in optimizing catalytic activity by contributing both to stabilizing enzyme structure and improving enzyme-substrate interaction. The N-Del mutant enzyme is considerably less efficient than the parent enzyme. The contribution of this domain cannot, on the basis of these data, be attributed to one particular amino acid but rather to a number of subtle interactions of amino acid residues in the domain as well as between the particular -lactam substrate and the adjacent subunit in the tetramer.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Pathology and Microbiology, School of Medical Sciences, University Walk, University of Bristol, Bristol, BS8 1TD UK. Tel.: 44-(0)117-928-7897; Fax: 44- (0)117-928-7896; E-mail: A.M.Simm@bristol.ac.uk.

Published, JBC Papers in Press, April 8, 2002, DOI 10.1074/jbc.M201524200

	ABBREVIATIONS

The abbreviations used are: N-Del, N-terminal deletion mutant L1 enzyme; AU, absorbance units; ELISA, enzyme-linked immunosorbent assay.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REACTION SCHEME 1 REFERENCES

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