Beta-lactam Substrates Research Articles

Involvement of the non-active site Residues in the Catalytic Activity of NDM-4 Metallo beta-lactamase.

The rise of New Delhi metallo beta-lactamase (NDM) producing bacteria imposes a significant threat to the treatment of bacterial infections due to their broad spectrum against beta-lactams. The activity of metallo beta-lactamases is affected by active site residues as well as residues near the active site. Therefore, we aimed to identify the amino acid residues around the active site of NDM-4 which influence its function. To achieve that, seven substitution mutations (S191A, D192A, S213A, K216A, S217A, D223A and D225A) of NDM-4 were generated through site-directed mutagenesis. Out of these, expression of NDM-4_D192A and NDM-4_S217A in Escherichia coli cells increased the beta-lactam susceptibility as compared to NDM-4. Further, proteins were purified to assess the effect of substitution mutations on zinc content, in vitro catalytic efficiency, and stability of NDM-4. The catalytic efficiency was reduced for these mutants (D192A and S217A) towards beta-lactam substrates, while the thermal stability remained insubstantial as compared to NDM-4. However, the purified NDM-4_D192A exhibited altered zinc content. In silico studies reveal that these changes might be the outcomes of alterations in hydrogen bonding networks and substrate interactions. Taken together, we infer that the D192 and the S217 residues play a substantial role in the activity of NDM-4.

New Beta-lactamases in Candidate Phyla Radiation: Owning Pleiotropic Enzymes Is a Smart Paradigm for Microorganisms with a Reduced Genome.

The increased exploitation of microbial sequencing methods has shed light on the high diversity of new microorganisms named Candidate Phyla Radiation (CPR). CPR are mainly detected via 16S rRNA/metabarcoding analyses or metagenomics and are found to be abundant in all environments and present in different human microbiomes. These microbes, characterized by their symbiotic/epiparasitic lifestyle with bacteria, are directly exposed to competition with other microorganisms sharing the same ecological niche. Recently, a rich repertoire of enzymes with antibiotic resistance activity has been found in CPR genomes by using an in silico adapted screening strategy. This reservoir has shown a high prevalence of putative beta-lactamase-encoding genes. We expressed and purified five putative beta-lactamase sequences having the essential domains and functional motifs from class A and class B beta-lactamase. Their enzymatic activities were tested against various beta-lactam substrates using liquid chromatography-mass spectrometry (LC-MS) and showed some beta-lactamase activity even in the presence of a beta-lactamase inhibitor. In addition, ribonuclease activity was demonstrated against RNA that was not inhibited by sulbactam and EDTA. None of these proteins could degrade single- and double-stranded-DNA. This study is the first to express and test putative CPR beta-lactamase protein sequences in vitro. Our findings highlight that the reduced genomes of CPR members harbor sequences encoding for beta-lactamases known to be multifunction hydrolase enzymes.

A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of DD-carboxypeptidase and beta-lactamase.

DD-carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the dd-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (ΔdacAdacC) of E. coli, strengthening its physiology as a dd-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours dd-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities.

Hydrolysis of Organophosphate Esters: Phosphotriesterase Activity of Metallo‐β‐lactamase and Its Functional Mimics

The phosphotriesterase (PTE) activity of a series of binuclear and mononuclear zinc(II) complexes and metallo-beta-lactamase (mbetal) from Bacillus cereus was studied. The binuclear complex 1, which exhibits good mbetal activity, shows poor PTE activity. In contrast, complex 2, a poor mimic of mbetal, exhibits much higher activity than 1. The replacement of Cl(-) ligands by OH(-) is important for the high PTE activity of complex 2 because this complex does not show any catalytic activity in methanol. The natural enzyme mbetal from B. cereus is also found to be an inefficient catalyst in the hydrolysis of phosphotriesters. These observations indicate that the binding of beta-lactam substrates at the binuclear zinc(II) center is different from that of phosphotriesters. Furthermore, phosphodiesters, the products from the hydrolysis of triesters, significantly inhibit the PTE activity of mbetal and its functional mimics. Although the mononuclear complexes 3 and 4 exhibited significant mbetal activity, these complexes are found to be almost inactive in the hydrolysis of phosphotriesters. These observations indicate that the elimination of phosphodiesters from the reaction site is important for the PTE activity of zinc(II) complexes.

Novel Beta-Lactam Antibiotics Derivatives: Their New Applications as Gene Reporters, Antitumor Prodrugs and Enzyme Inhibitors

Since the antibiotic properties of penicillin were first noticed in the beginning of last century, beta-lactam based antibiotics have been well developed as miracle drugs for the therapy of bacterial infectious diseases in clinics. Recently, these "old" antibiotics and their relevant derivatives have also found new applications as gene reporters, anti-cancer prodrugs and enzyme inhibitors. In this review, we will introduce the latest developments in the study of these new applications based on literatures reported over the last decade. The first section covers the recent developments of beta-lactam antibiotics as drugs against bacteria, the second section briefly discusses the occurrence of bacterial resistance and mechanistic studies of beta-lactam resistance in bacteria, the third section presents the current development of fluorogenic cephalosporin based beta-lactam probes for real-time imaging of gene expression, and the fourth section describes relevant studies on beta-lactam based substrates as anti-tumor prodrugs. Beta-lactam substrates as protease inhibitors will be also described in the fifth section. The final section summarizes future perspectives for beta-lactam antibiotic derivatives as scaffolds in the fields of molecular imaging, drug delivery and enzymatic assays.

A complete library of amino acid alterations at R306 in Streptomyces clavuligerus deacetoxycephalosporin C synthase demonstrates its structural role in the ring‐expansion activity

In a previous study, the conserved arginine residue at position 306 of Streptomyces clavuligerus deacetoxycephalsoporin C synthase (scDAOCS), when mutated to leucine, resulted in 191% increase in converting ampicillin to its expanded cephalosporin moiety compared with that of the wild-type enzyme. However, the role of this residue in eliciting the improved enzymatic activity is not well understood. In this study, probing the molecular basis of amino acid substitutions at position 306 has underscored its importance for engineering various improvements in the ring expansion activity. Structural modeling using SwissPdbViewer revealed that R306 is surrounded by a hydrophobic cleft formed by residues Y184, L186, W297, I298, and V303. Hence, the improved activity achieved by the R306L mutation was probably because of better hydrophobic packing in this region. To evaluate the role of amino acids at position 306 of scDAOCS and its influence on the molecular status of the enzyme at this locality, alteration to 18 other amino acids was done by site-directed mutagenesis. The effects of each substitution on the enzyme activity were determined by bioassay using penicillin substrates: ampicillin, penicillin G, phenethicillin, and carbenicillin. Results obtained showed a drastic reduction in enzyme activity when R306 was replaced with charged or polar residues, thus emphasizing the importance of hydrophobic packing around this site. The bioassay results also illustrated that apart from leucine, substitutions to nonpolar residues, isoleucine and methionine, were able to improve the ampicillin conversion activity of scDAOCS by 168 and 113% of the wild-type enzyme activity, respectively. Similar trend of effects from each mutation was also observed for penicillin G, phenethicillin, and carbenicillin conversions. The enhanced enzyme activities were supported by spectrophotometric assay indicating that all these mutants have lower K(m) values (R306L: 1.09 mM; R306I: 2.64 mM; R306M: 5.68 mM) than the wild-type enzyme (8.33 mM), resulting in improvement in the enzyme's substrate binding affinity. Hence, this mutational study of amino acids situated at 306 of scDAOCS has provided a better understanding of the significance of specific amino acid residues at this position which can improve its ring-expansion activity when given a plethora of beta-lactam substrates to generate corresponding, possibly new, cephalosporins.

Antibiotic Resistance: Mono‐ and Dinuclear Zinc Complexes as Metallo‐β‐Lactamase Mimics

Biomimetic systems containing one or two zinc(II) ions supported by phenolate ligands were developed as functional mimics of metallo-beta-lactamase. These complexes were shown to catalytically hydrolyze beta-lactam substrates, such as oxacillin and penicillin G. The dinuclear zinc complex 1, which has a coordinated water molecule, exhibits high beta-lactamase activity, whereas the dinuclear zinc complex 2, which has no water molecules, but labile chloride ligands, shows a much lower activity. The high beta-lactamase activity of complex 1 can be ascribed to the presence of a zinc-bound water molecule that is activated by being hydrogen bonded to acetate substituents. The kinetics of the hydrolysis of oxacillin by complex 1 and the effect of pH on the reaction rates are reported in detail. In addition, the kinetic parameters obtained for the synthetic analogues are compared with those of the natural metallo-beta-lactamase from Bacillus cereus (BcII). To understand the role of the second metal ion in hydrolysis, the syntheses and catalytic activities of two mononuclear complexes (3 and 4) that include coordinated water molecules are described. Interestingly, the mononuclear zinc complexes 3 and 4 also exhibit high activity, supporting the assumption that the second zinc ion is not crucial for the beta-lactamase activity.

Antibiotic Recognition by Binuclear Metallo-β-Lactamases Revealed by X-ray Crystallography

Metallo-beta-lactamases are zinc-dependent enzymes responsible for resistance to beta-lactam antibiotics in a variety of host bacteria, usually Gram-negative species that act as opportunist pathogens. They hydrolyze all classes of beta-lactam antibiotics, including carbapenems, and escape the action of available beta-lactamase inhibitors. Efforts to develop effective inhibitors have been hampered by the lack of structural information regarding how these enzymes recognize and turn over beta-lactam substrates. We report here the crystal structure of the Stenotrophomonas maltophilia L1 enzyme in complex with the hydrolysis product of the 7alpha-methoxyoxacephem, moxalactam. The on-enzyme complex is a 3'-exo-methylene species generated by elimination of the 1-methyltetrazolyl-5-thiolate anion from the 3'-methyl group. Moxalactam binding to L1 involves direct interaction of the two active site zinc ions with the beta-lactam amide and C4 carboxylate, groups that are common to all beta-lactam substrates. The 7beta-[(4-hydroxyphenyl)malonyl]-amino substituent makes limited hydrophobic and hydrogen bonding contacts with the active site groove. The mode of binding provides strong evidence that a water molecule situated between the two metal ions is the most likely nucleophile in the hydrolytic reaction. These data suggest a reaction mechanism for metallo-beta-lactamases in which both metal ions contribute to catalysis by activating the bridging water/hydroxide nucleophile, polarizing the substrate amide bond for attack and stabilizing anionic nitrogen intermediates. The structure illustrates how a binuclear zinc site confers upon metallo-beta-lactamases the ability both to recognize and efficiently hydrolyze a wide variety of beta-lactam substrates.

Correlation between Catalytic Efficiency and the Transcription Read-Out in Chemical Complementation: A General Assay for Enzyme Catalysis

High-throughput assays for enzyme catalysis that can be applied to a broad range of chemical reactions are key to advances in directed evolution and proteomics. Recently, we reported such a general assay, chemical complementation, which links enzyme catalysis to reporter gene transcription in vivo using the yeast three-hybrid assay. In this proof-of-principle experiment, it was shown that a wild-type beta-lactamase enzyme could be isolated from a pool of inactive mutants using a lacZ screen. Ideally, however, such an assay should be able to distinguish enzymes based on their catalytic activity. Thus, here, we set out to determine if the catalytic efficiency of an enzyme variant does in fact correlate with its level of transcription activation in the chemical complementation assay. First, the reaction mechanism for the cleavage of the beta-lactam substrate used in the chemical complementation proof-of-principle experiment was determined. Then a series of beta-lactamase variants was designed to span several orders of magnitude in k(cat)/K(m). The activity of each variant was determined both in vitro using purified enzyme and in vivo in the chemical complementation transcription assay. Beta-lactamase variants spanning three-orders of magnitude in k(cat)/K(m) could be distinguished in the assay, and the catalytic efficiency of each variant correlated with its level of transcription activation in vivo. These results establish the suitability of chemical complementation for the directed evolution of enzymes with improvements in catalytic activity and for profiling the relative substrate specificities of a group of enzymes in proteomics applications.

A dynamic structure for the acyl-enzyme species of the antibiotic aztreonam with the Citrobacter freundii beta-lactamase revealed by infrared spectroscopy and molecular dynamics simulations.

Infrared difference spectra show that at least 4 conformations coexist for the ester carbonyl group of the stable acyl-enzyme species formed between the antibiotic aztreonam and the class C beta-lactamase from Citrobacter freundii. A novel method for the assignment of the bands that arise from the ester carbonyl group has been employed. This has made use of the finding that the infrared absorption intensity of aliphatic esters is surprisingly constant, so a direct comparison with simple model esters has been possible. This has allowed a clear distinction to be made between ester and amide (protein) absorptions. The polarity of the conformer environment varies from hexane-like to strongly hydrogen-bonded. We assume that the conformer with the lowest frequency (1,690 cm(-)(1)) and hence the strongest hydrogen-bonding is the singular conformer observed in the X-ray crystallographic structure, since a good interaction via two hydrogen bonds with the oxyanion hole is seen. Molecular dynamics simulation by the method of locally enhanced sampling revealed that the motion of the ester carbonyl of the acyl-enzyme species in and out of the oxyanion hole is facile. The simulation revealed two pathways for this motion that would go through intermediates that first break one or the other of the two hydrogen bonds to the oxyanion hole, prior to departure of the carbonyl moiety out of the active site. It is likely that such motion for the acyl-enzyme species might also occur with more typical beta-lactam substrates for beta-lactamases, but their detection in the more rapid time scale may prove a challenge.

Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum beta-lactamase CTX-M-15 and of its structurally related beta-lactamase CTX-M-3.

The extended-spectrum beta-lactamase CTX-M-15 confers resistance to ceftazidime, unlike the majority of CTX-M-type enzymes. Kinetic parameters were determined from purified CTX-M-15 and CTX-M-3, which differ by the single amino acid substitution Asp-240 to Gly, according to the Ambler numbering of class A beta-lactamases. Relative molecular masses of CTX-M-15 and CTX-M-3 were approximately 29 kDa and pI values were 8.9 and 8.4, respectively. CTX-M-15 had higher affinities for beta-lactams (lower K(m) values) than those of CTX-M-3 but catalytic efficiency (k(cat)/K(m) values) was variable depending on the beta-lactam substrate. Only CTX-M-15 showed a measurable catalytic efficiency for ceftazidime. Clavulanic acid and tazobactam were good inhibitors of both enzymes. MICs of beta-lactams for Escherichia coli reference strains expressing cloned beta-lactamase genes in the same genetic background were similar except for ceftazidime. This work underlines the fact that some CTX-M enzymes may hydrolyse ceftazidime and thus confer resistance to this expanded-spectrum cephalosporin in Enterobacteriaceae.

Directed evolution of a glutaryl acylase into an adipyl acylase.

Semi-synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs, and are produced from 7-aminodesacetoxycephalosporanic acid (7-ADCA). Recently new routes have been developed which allow for the production of adipyl-7-ADCA by a novel fermentation process. To complete the biosynthesis of 7-ADCA a highly active adipyl acylase is needed for deacylation of the adipyl derivative. Such an adipyl acylase can be generated from known glutaryl acylases. The glutaryl acylase of Pseudomonas SY-77 was mutated in a first round by exploration mutagenesis. For selection the mutants were grown on an adipyl substrate. The residues that are important to the adipyl acylase activity were identified, and in a second round saturation mutagenesis of this selected stretch of residues yielded variants with a threefold increased catalytic efficiency. The effect of the mutations could be rationalized on hindsight by the 3D structure of the acylase. In conclusion, the substrate specificity of a dicarboxylic acid acylase was shifted towards adipyl-7-ADCA by a two-step directed evolution strategy. Although derivatives of the substrate were used for selection, mutants retained activity on the beta-lactam substrate. The strategy herein described may be generally applicable to all beta-lactam acylases.

Antimicrobial Resistance Surveillance of Gram-Negative Bacteria Isolated from Intensive Care Units of Four Different Hospitals in Turkey

Background: The aim of this study was to determine the antimicrobial resistance of gram-negative rods (GNRs) isolated from surgical intensive care units and to establish the prevalence of extended-spectrum beta-lactamases (ESBLs) and inducible beta-lactamases (IBLs). We also wished to determine the widespread beta-lactam substrate in ESBL-positive GNRs by two different E-test strips and to discuss the value of the routine utilization of these substrates together. Methods: Out of 348 nosocomial gram-negative strains isolated with similar methods, 236 strains with resistance to the beta-lactam group of antimicrobials using the E-test method were included in this study. Two different strips were used for the detection of ESBLs: cefotaxime/cefotaxime + clavulanic acid (CT/CTL) and ceftazidime/ceftazidime + clavulanic acid (TZ/TZL). For IBLs, the double-disk method was used. Results: The order of frequency of the strains, starting with the most frequent, was Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. In the 236 strains, the ESBL positivity rate was found to be 19.5% with TZ/TZL and CT/CTL strips, while it was 13.2% for IBL in 348 strains. Seventy-one percent of ESBL-positive strains gave parallel results with TZ/TZL and CT/CTL. ESBL positivity with only TZ/TZL or only CT/CTL was found to be 18 and 8%. Conclusions: Our study showed that except for imipenem, amikacin and ciprofloxacin, there was a high resistance to other antimicrobials, and multiresistance rates were increased in the strains in which ESBLs and IBLs were detected. In particular, the increasing prevalence of ESBLs in K. pneumoniae and IBLs in P. aeruginosa emphasizes the importance of the problem of infection control and antibiotic administration policies. Although it was seen that the prevalence of substrate templates in the detection of ESBL positivity was similar, we think that it is more useful to use two different strips together to obtain precise results.

Molecular dynamics simulations of the mononuclear zinc-beta-lactamase from Bacillus cereus complexed with benzylpenicillin and a quantum chemical study of the reaction mechanism.

Herein, we present results from MD simulations of the Michaelis complex formed between the B. cereus zinc-beta-lactamase enzyme and benzylpenicillin. The structural and dynamical effects induced by substrate-binding, the specific role of the conserved residues, and the near attack conformers of the Michaelis complex are discussed. Quantum chemical methods (HF/6-31G* and B3LYP/6-31G*) are also applied to study the hydrolysis reaction of N-methylazetidinone catalyzed by a monozinc system consisting of the side chains of the histidine residues (His86, His88, and His149) complexed with Zn-OH and the side chains of Asp90 and His210. From this model system, we built molecular-mechanics representations of the prereactive complex and transition state configurations docked into the active site. Linear-scaling semiempirical calculations coupled with a continuum solvent model were then performed on these static models. We propose that the experimental rate data for the B. cereus enzyme is compatible with a one-step mechanism for the hydrolysis of beta-lactam substrates in which His210 acts as a proton donor.

Novel Mechanism of Hydrolysis of Therapeutic β-Lactams byStenotrophomonas maltophilia L1 Metallo-β-lactamase

Stopped-flow tryptophan fluorescence under single turnover and pseudo-first-order conditions has been used to investigate the kinetic mechanism of beta-lactam hydrolysis by the Stenotrophomonas maltophilia L1 metallo-beta-lactamase. For the cephalosporin substrates nitrocefin and cefaclor and the carbapenem meropenem, a substantial quench of fluorescence is observed on association of substrate with enzyme. We have assigned this to a rearrangement event subsequent to formation of an initial collision complex. For the colorimetric compound nitrocefin, decay of this dark inter- mediate represents the overall rate-determining step for the reaction and is equivalent to decay of a previously observed state in which the beta-lactam amide bond has already been cleaved. For both cefaclor and meropenem, the rate-determining step for hydrolysis is loss of a second, less quenched state, in which, however, the beta-lactam amide bond remains intact. We suggest, therefore, that the mechanism of hydrolysis of nitrocefin by binuclear metallo-beta-lactamases may be atypical and that cleavage of the beta-lactam amide bond is the rate-determining step for breakdown of the majority of beta-lactam substrates by the L1 enzyme.

Structures of ceftazidime and its transition-state analogue in complex with AmpC beta-lactamase: implications for resistance mutations and inhibitor design.

Third-generation cephalosporins are widely used beta-lactam antibiotics that resist hydrolysis by beta-lactamases. Recently, mutant beta-lactamases that rapidly inactivate these drugs have emerged. To investigate why third-generation cephalosporins are relatively stable to wild-type class C beta-lactamases and how mutant enzymes might overcome this, the structures of the class C beta-lactamase AmpC in complex with the third-generation cephalosporin ceftazidime and with a transition-state analogue of ceftazidime were determined by X-ray crystallography to 2.0 and 2.3 A resolution, respectively. Comparison of the acyl-enzyme structures of ceftazidime and loracarbef, a beta-lactam substrate, reveals that the conformation of ceftazidime in the active site differs from that of substrates. Comparison of the structures of the acyl-enzyme intermediate and the transition-state analogue suggests that ceftazidime blocks formation of the tetrahedral transition state, explaining why it is an inhibitor of AmpC. Ceftazidime cannot adopt a conformation competent for catalysis due to steric clashes that would occur with conserved residues Val211 and Tyr221. The X-ray crystal structure of the mutant beta-lactamase GC1, which has improved activity against third-generation cephalosporins, suggests that a tandem tripeptide insertion in the Omega loop, which contains Val211, has caused a shift of this residue and also of Tyr221 that would allow ceftazidime and other third-generation cephalosporins to adopt a more catalytically competent conformation. These structural differences may explain the extended spectrum activity of GC1 against this class of cephalosporins. In addition, the complexed structure of the transition-state analogue inhibitor (K(i) 20 nM) with AmpC reveals potential opportunities for further inhibitor design.

Intermediate in beta-lactam hydrolysis catalyzed by a dinuclear zinc(II) complex: relevance to the mechanism of metallo-beta-lactamase.

Inactivation of beta-lactam antibiotics by metallo-beta-lactamase enzymes is a well-recognized pathway of antibiotic resistance in bacteria. As part of extensive mechanistic studies, the hydrolysis of a beta-lactam substrate nitrocefin (1) catalyzed by dinuclear zinc(II) model complexes was investigated in nonaqueous solutions. The initial step involves monodentate coordination of the nitrocefin carboxylate group to the dizinc center. The coordinated substrate is then attacked intramolecularly by the bridging hydroxide to give a novel intermediate (2') characterized by its prominent absorbance maximum at 640 nm, which affords a blue color. The NMR and IR spectroscopic data of 2' are consistent with it being zinc(II)-bound N-deprotonated hydrolyzed nitrocefin that forms from the tetrahedral intermediate upon C-N bond cleavage. Protonation of the leaving group is the rate-limiting step in DMSO solution and occurs after the C-N bond-breaking step. Addition of strong acids results in rapid conversion of 2' into hydrolyzed nitrocefin (3). The latter can be converted back to the blue species (2') upon addition of base. The low pK(a) value for the amino group in hydrolyzed nitrocefin is explained by its involvement in extended conjugation and by coordination to zinc(II). The blue intermediate (2') in the model system resembles well that in the enzymatic system, judging by its optical properties. The greater stability of the intermediate in the model, however, allowed its characterization by (13)C NMR and infrared, as well as electronic, spectroscopy.

Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site.

Beta-lactamases hydrolyze beta-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. beta-Lactams that contain a bulky 6(7)alpha substituent, such as imipenem and moxalactam, actually inhibit serine beta-lactamases and are widely used for this reason. Although mutant serine beta-lactamases have arisen that hydrolyze beta-lactamase resistant beta-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine beta-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7)alpha substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C beta-lactamases (Asn132 in class A beta-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C beta-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 --> Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 A. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces beta-lactams with 6(7)alpha substituents out of a catalytically competent configuration, making them inhibitors, the residue is essential for orienting beta-lactam substrates and cannot simply be replaced with a much smaller residue to restore catalytic activity. Designing beta-lactam inhibitors that interact unfavorably with this conserved residue when in the covalent adduct merits further investigation.

Characterization of the metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97 reveals the existence of bla(IMP) allelic variants carried by gene cassettes of different phylogeny.

The metallo-beta-lactamase determinant of Acinetobacter baumannii AC-54/97, a clinical isolate from Italy that was previously shown to produce an enzyme related to IMP-1, was isolated by means of a PCR methodology which targets amplification of gene cassette arrays inserted into class 1 integrons. Sequencing revealed that this determinant was an allelic variant (named bla(IMP-2)) of bla(IMP) found in Japanese isolates and that it was divergent from the latter by 12% of its nucleotide sequence, which evidently had been acquired independently. Similar to bla(IMP), bla(IMP-2) was also carried by an integron-borne gene cassette. However, the 59-base element of the bla(IMP-2) cassette was unrelated to those of the bla(IMP) cassettes found in Japanese isolates, indicating a different phylogeny for the gene cassettes carrying the two allelic variants. Expression of the integron-borne bla(IMP-2) gene in Escherichia coli resulted in a significant decrease in susceptibility to a broad array of beta-lactams (ampicillin, carbenicillin, cephalothin, cefoxitin, ceftazidime, cefepime, and carbapenems). The IMP-2 enzyme was purified from an Escherichia coli strain carrying the cloned determinant, and kinetic parameters were determined with several beta-lactam substrates. Compared to IMP-1, the kinetic parameters of IMP-2 were similar overall with some beta-lactam substrates (cefoxitin, ceftazidime, cefepime, and imipenem) but remarkably different with others (ampicillin, carbenicillin, cephaloridine, and meropenem), revealing a functional significance of at least some of the mutations that differentiate the two IMP variants. Present findings suggest that the environmental reservoir of bla(IMP) alleles could be widespread and raise a question about the global risk of their transfer to clinically relevant species.

PKa calculations for class A beta-lactamases: influence of substrate binding.

Beta-Lactamases are responsible for bacterial resistance to beta-lactams and are thus of major clinical importance. However, the identity of the general base involved in their mechanism of action is still unclear. Two candidate residues, Glu166 and Lys73, have been proposed to fulfill this role. Previous studies support the proposal that Glu166 acts during the deacylation, but there is no consensus on the possible role of this residue in the acylation step. Recent experimental data and theoretical considerations indicate that Lys73 is protonated in the free beta-lactamases, showing that this residue is unlikely to act as a proton abstractor. On the other hand, it has been proposed that the pKa of Lys73 would be dramatically reduced upon substrate binding and would thus be able to act as a base. To check this hypothesis, we performed continuum electrostatic calculations for five wild-type and three beta-lactamase mutants to estimate the pKa of Lys73 in the presence of substrates, both in the Henri-Michaelis complex and in the tetrahedral intermediate. In all cases, the pKa of Lys73 was computed to be above 10, showing that it is unlikely to act as a proton abstractor, even when a beta-lactam substrate is bound in the enzyme active site. The pKa of Lys234 is also raised in the tetrahedral intermediate, thus confirming a probable role of this residue in the stabilization of the tetrahedral intermediate. The influence of the beta-lactam carboxylate on the pKa values of the active-site lysines is also discussed.