OHIO-1 beta-lactamase mutants: the Arg244Ser mutant and resistance to beta-lactams and beta-lactamase inhibitors.

Mutations at residue 244 (Ambler numbering system) in the class A TEM beta-lactamase confer resistance to inactivation by beta-lactamase inhibitors and result in diminished turnover of beta-lactam substrates. The Arg244Ser mutant of the OHIO-1 beta-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to beta-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5alpha expressing the Arg244Ser beta-lactamase were reduced when compared to the strain bearing the OHIO-1 beta-lactamase: ampicillin, 512 vs. 8192 micrograms ml-1; cephaloridine, 4 vs. 32 micrograms ml-1, respectively. The MICs for the beta-lactam beta-lactamase inhibitor combinations demonstrated resistance only to ampicillin-clavulanate, 16/8 vs. 8/4 micrograms ml-1 respectively. In contrast, there was increased susceptibility to ampicillin-sulbactam, ampicillin-tazobactam, and piperacillin-tazobactam. When compared to the OHIO-1 beta-lactamase homogenous preparations of the Arg244Ser beta-lactamase enzyme demonstrated increased Km and decreased kcat values for benzylpenicillin (Km=17 vs. 50 microM, kcat=345 vs. 234 s-1) and cephaloridine (Km=97 vs. 202 microM, kcat=1023 vs. 202 s-1). Although the Ki and IC50 values were increased for each inhibitor when compared to OHIO-1 beta-lactamase, the turnover numbers (tn) required for inactivation were increased only for clavulanate. For the Arg244Ser mutant enzyme of OHIO-1, the increased Ki, decreased tn for the sulfones, and different partition ratio (kcat/kinact) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A beta-lactamase enzymes may react differently with identical substitutions in structurally conserved amino acids. The resistance phenotype of a specific mutations can vary depending on the enzyme.     

Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130

Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.     

Beta-ketophosphonates as beta-lactamase inhibitors: Intramolecular cooperativity between the hydrophobic subsites of a class D beta-lactamase

A series of aryl and arylmethyl beta-aryl-beta-ketophosphonates have been prepared as potential beta-lactamase inhibitors. These compounds, as fast, reversible, competitive inhibitors, were most effective (micromolar K(i) values) against the class D OXA-1 beta-lactamase but had less activity against the OXA-10 enzyme. They were also quite effective against the class C beta-lactamase of Enterobacter cloacae P99 but less so against the class A TEM-2 enzyme. Reduction of the keto group to form the corresponding beta-hydroxyphosphonates led to reduced inhibitory activity. Molecular modeling, based on the OXA-1 crystal structure, suggested interaction of the aryl groups with the hydrophobic elements of the enzyme's active site and polar interaction of the keto and phosphonate groups with the active site residues Ser 115, Lys 212 and Thr 213 and with the non-conserved Ser 258. Analysis of binding free energies showed that the beta-aryl and phosphonate ester aryl groups interacted cooperatively within the OXA-1 active site. Overall, the results suggest that quite effective inhibitors of class C and some class D beta-lactamases could be designed, based on the beta-ketophosphonate platform.     

Structure, function, and fate of the BlaR signal transducer involved in induction of beta-lactamase in Bacillus licheniformis.

The membrane-spanning protein BlaR is essential for the induction of beta-lactamase in Bacillus licheniformis. Its nature and location were confirmed by the use of an antiserum specific for its carboxy-terminal penicillin sensor, its function was studied by genetic dissection, and the structure of the penicillin sensor was derived from hydrophobic cluster analysis of the amino acid sequence by using, as a reference, the class A beta-lactamases with known three-dimensional structures. During the first 2 h after the addition of the beta-lactam inducer, full-size BlaR, bound to the plasma membrane, is produced, and then beta-lactamase is produced. By 2 h after induction, BlaR is present in various (membrane-bound and cytosolic) forms, and there is a gradual decrease in beta-lactamase production. The penicillin sensors of BlaR and the class D beta-lactamases show strong similarities in primary structures. They appear to have the same basic spatial disposition of secondary structures as that of the class A beta-lactamases, except that they lack several alpha helices and, therefore, have a partially uncovered five-stranded beta sheet and a more readily accessible active site. Alterations of BlaR affecting conserved secondary structures of the penicillin sensor and specific sites of the transducer annihilate beta-lactamase inducibility.     

Characterization of the membrane beta-lactamase in Bacillus cereus 569/H/9

The membrane-bound beta-lactamase from Bacillus cereus, strain 569/H/9, has been purified to apparent homogeneity. Nonionic detergent (0.5% Triton X-100) is required to keep the enzyme (traditionally called gamma-penicillinase and now called beta-lactamase III) in solution. Antibodies to beta-lactamase III have been prepared, and the membrane-bound enzyme is immunochemically distinct from the extracellular enzymes. beta-Lactamase III has a molecular weight of 31 500, in contrast to the extracellular enzymes beta-lactamase I and beta-lactamase II which have molecular weights of 30 000 and 22 000, respectively. The isoelectric point of beta-lactamase III is pH 6.8, whereas beta-lactamase I and beta-lactamase II have isoelectric points about 8.6 and 8.3. The amino acid composition of beta-lactamase III differs from those of beta-lactamase I and beta-lactamase II; however, the difference index between the compositions of beta-lactamase I and beta-lactamase III (52%) suggests relatedness. beta-Lactamase III is inactivated by 6 beta-bromopenicillanic acid and by the sulfone of 6 alpha-chloropenicillanic acid, and cephalosporins are poorer substrates than penicillins. beta-Lactamase III may be a membrane-bound class A beta-lactamase.     

Characterization of an extended-spectrum class C beta-lactamase of Citrobacter freundii

Citrobacter freundii GC3 is a clinical isolate which showed moderate resistance to oxyimino beta-lactams such as ceftazidime and aztreonam. This drug resistance was due to an extended-spectrum class C beta-lactamase encoded by chromosomal gene(s). The GC3 beta-lactamase showed high amino acid sequence homology to a known C. freundii beta-lactamase, i.e., 346 of 361 amino acids were identical with those of C. freundii GN346 beta-lactamase (Tsukamoto, K. et al, Eur. J. Biochem. 188, 15-22, 1990). Asp198 was the only dissimilar amino acid found in the omega loop region, known as the hot spot for extended-spectrum resistance in class C beta-lactamases (Haruta, S. et al, Microbiol. Immunol. 42, 165-169, 1998). However, Asp198 was eliminated as a cause of the extended-spectrum resistance by the substitution of Asn for Asp198. Subsequent investigation suggested that the moderate resistance to oxyimino beta-lactams is attributable to the replacement of amino acids on the enzyme's surface area, far from the active-site. Some or all of the replacements are assumed to delicately modify the active-site configuration. The GC3 beta-lactamase is the first example of an extended-spectrum class C beta-lactamase in which mutations are independent of the omega loop.     

Structural basis for the extended substrate spectrum of CMY-10, a plasmid-encoded class C beta-lactamase

The emergence and dissemination of extended-spectrum (ES) beta-lactamases induce therapeutic failure and a lack of eradication of clinical isolates even by third-generation beta-lactam antibiotics like ceftazidime. CMY-10 is a plasmid-encoded class C beta-lactamase with a wide spectrum of substrates. Unlike the well-studied class C ES beta-lactamase from Enterobacter cloacae GC1, the Omega-loop does not affect the active site conformation and the catalytic activity of CMY-10. Instead, a three-amino-acid deletion in the R2-loop appears to be responsible for the ES activity of CMY-10. According to the crystal structure solved at 1.55 A resolution, the deletion significantly widens the R2 active site, which accommodates the R2 side-chains of beta-lactam antibiotics. This observation led us to demonstrate the hydrolysing activity of CMY-10 towards imipenem with a long R2 substituent. The forced mutational analyses of P99 beta-lactamase reveal that the introduction of deletion mutations into the R2-loop is able to extend the substrate spectrum of class C non-ES beta-lactamases, which is compatible with the isolation of natural class C ES enzymes harbouring deletion mutations in the R2-loop. Consequently, the opening of the R2 active site by the deletion of some residues in the R2-loop can be considered as an operative molecular strategy of class C beta-lactamases to extend their substrate spectrum.     

Design, synthesis, and evaluation of alpha-ketoheterocycles as class C beta-lactamase inhibitors

A series of specific alpha-ketoheterocycles (benzoxazole, thiazole, imidazole, tetrazole, and thiazole-4-carboxylate) has been synthesized in order to assess their potential as beta-lactamase inhibitors. The syntheses were achieved either by construction of the heterocycle (benzoxazole) from an appropriate alpha-hydroxyimidate, followed by oxidation of the alcohol, or by direct reaction of methyl phenaceturate with a lithiated heterocycle. The properties of these compounds in aqueous solution are described and their inhibitory activity against beta-lactamases assessed. They did inhibit the class C beta-lactamase of Enterobacter cloacae P99 but not the TEM beta-lactamase. The most effective inhibitor of the former enzyme (K(i)=0.11 mM) was 5-(phenylacetylglycyl) tetrazole, probably because it is an anion at neutral pH. Interpretation of the results was aided by computational models of the tetrahedral adducts. Most of the compounds also inhibited alpha-chymotrypsin but not porcine pancreatic elastase.     

Synthesis and beta-lactamase reactivity of alpha-substituted phenaceturates

Beta-lactams with 6alpha (penicillins) or 7alpha (cephalosporins) substituents are often beta-lactamase inhibitors. This paper assesses the effect of such substituents on acyclic beta-lactamase substrates. Thus, a series of m-carboxyphenyl phenaceturates, substituted at the glycyl alpha-carbon by -OMe, -CH(2)OH, -CO(2)(-), and -CH(2)NH(3)(+), have been prepared, and tested for their reactivity against serine beta-lactamases. The latter two are novel substituents in beta-lactamase substrates. The methoxy and hydroxymethyl compounds were found to be poor to moderately good substrates, depending on the enzyme. The aminomethyl compound gave rise to a transiently stable (t(1/2)=4.6s) complex on its reaction with a class C beta-lactamase. The reactivity of the compounds against three low molecular weight DD-peptidases was also tested. Again, the methoxy and hydroxymethyl compounds proved to be quite good substrates with no sign of inhibitory complexes. The DD-peptidases reacted with one enantiomer (the compounds were prepared as racemates), presumably the D compound. The class C beta-lactamase reacted with both D and L enantiomers although it preferred the latter. The structural bases of these stereo-preferences were explored by reference to the crystal structure of the enzyme by molecular modeling studies. The aminomethyl compound was unreactive with the DD-peptidases, whereas the carboxy compound did not react with any of the above-mentioned enzymes. The inhibitory effects of the -OMe and -CH(2)OH substituents in beta-lactams apparently require a combination of the substituent and the pendant leaving group of the beta-lactam at the acyl-enzyme stage.     

Inactivation of CMY-2 beta-lactamase by tazobactam: initial mass spectroscopic characterization.

The CMY-2 beta-lactamase, a plasmid determined class C cephalosporinase, was shown to be susceptible to inhibition by tazobactam (K(i)=40 microM). The reaction product(s) of CMY-2 beta-lactamase with the beta-lactamase inhibitor tazobactam were analyzed by electrospray ionization/mass spectrometry (ESI/MS) to characterize the prominent intermediates of the inactivation pathway. The ESI/MS determined mass of CMY-2 beta-lactamase was 39851+/-3 Da. After inactivating CMY-2 beta-lactamase with excess tazobactam, a single species, M(r)=39931+/-3.0, was detected. Comparison of the peptide maps from tryptic digestion of the native enzyme and the inactivated beta-lactamase followed by LC/MS identified two 22 amino acid peptides containing the active site Ser64 modified by a fragment of tazobactam. These two peptides were increased in mass by 70 and 88 Da, respectively. UV difference spectra following inactivation revealed the presence of a new species with a 302 nm lambda(max). Based upon the increase in molecular mass of the tazobactam inactivated CMY-2 beta-lactamase, we propose that during the inactivation of this beta-lactamase by tazobactam an imine is formed. Tautomerization forms the spectrally observed enamine. Hydrolysis generates the covalently attached malonyl semialdehyde, its hydrate, or an enol. This work provides information on the mass of a stable enzyme intermediate of a class C beta-lactamase inactivated by tazobactam and, for the first time, unequivocal evidence that a cross-linked species is not required for apparent inactivation.     

Design of potent beta-lactamase inhibitors by phage display of beta-lactamase inhibitory protein

Beta-lactamase inhibitory protein (BLIP) binds tightly to several beta-lactamases including TEM-1 beta-lactamase (K(i) 0.1 nm). The TEM-1 beta-lactamase/BLIP co-crystal structure indicates that two turn regions in BLIP insert into the active site of beta-lactamase to block the binding of beta-lactam antibiotics. Residues from each turn, Asp(49) and Phe(142), mimic interactions made by penicillin G when bound in the beta-lactamase active site. Phage display was used to determine which residues within the turn regions of BLIP are critical for binding TEM-1 beta-lactamase. The sequences of a set of functional mutants from each library indicated that a few sequence types were predominant. These BLIP mutants exhibited K(i) values for beta-lactamase inhibition ranging from 0.01 to 0.2 nm. The results indicate that even though BLIP is a potent inhibitor of TEM-1 beta-lactamase, the wild-type sequence of the active site binding region is not optimal and that derivatives of BLIP that bind beta-lactamase extremely tightly can be obtained. Importantly, all of the tight binding BLIP mutants have sequences that would be predicted theoretically to form turn structures.     

Determinants of binding affinity and specificity for the interaction of TEM-1 and SME-1 beta-lactamase with beta-lactamase inhibitory protein

The hydrolysis of beta-lactam antibiotics by class A beta-lactamases is a common cause of bacterial resistance to these agents. The beta-lactamase inhibitory protein (BLIP) is able to bind and inhibit several class A beta-lactamases, including TEM-1 beta-lactamase and SME-1 beta-lactamase. Although the TEM-1 and SME-1 enzymes share 33% amino acid sequence identity and a similar fold, they differ substantially in surface electrostatic properties and the conformation of a loop-helix region that BLIP binds. Alanine-scanning mutagenesis was performed to identify the residues on BLIP that contribute to its binding affinity for each of these enzymes. The results indicate that the sequence requirements for binding are similar for both enzymes with most of the binding free energy provided by two patches of aromatic residues on the surface of BLIP. Polar residues such as several serines in the interface do not make significant contributions to affinity for either enzyme. In addition, the specificity of binding is significantly altered by mutation of two charged residues, Glu73 and Lys74, that are buried in the structure of the TEM-1.BLIP complex as well as by residues located on two loops that insert into the active site pocket. Based on the results, a E73A/Y50A double mutant was constructed that exhibited a 220,000-fold change in binding specificity for the TEM-1 versus SME-1 enzymes.     

Identification of residues in beta-lactamase critical for binding beta-lactamase inhibitory protein

beta-Lactamase inhibitory protein (BLIP) is a potent inhibitor of several beta-lactamases including TEM-1 beta-lactamase (Ki = 0.1 nM). The co-crystal structure of TEM-1 beta-lactamase and BLIP has been solved, revealing the contact residues involved in the interface between the enzyme and inhibitor. To determine which residues in TEM-1 beta-lactamase are critical for binding BLIP, the method of monovalent phage display was employed. Random mutants of TEM-1 beta-lactamase in the 99-114 loop-helix and 235-240 B3 beta-strand regions were displayed as fusion proteins on the surface of the M13 bacteriophage. Functional mutants were selected based on the ability to bind BLIP. After three rounds of enrichment, the sequences of a collection of functional beta-lactamase mutants revealed a consensus sequence for the binding of BLIP. Seven loop-helix residues including Asp-101, Leu-102, Val-103, Ser-106, Pro-107, Thr-109, and His-112 and three B3 beta-strand residues including Ser-235, Gly-236, and Gly-238 were found to be critical for tight binding of BLIP. In addition, the selected beta-lactamase mutants A113L/T114R and E240K were found to increase binding of BLIP by over 6- and 11-fold, respectively. Combining these substitutions resulted in 550-fold tighter binding between the enzyme and BLIP with a Ki of 0.40 pM. These results reveal that the binding between TEM-1 beta-lactamase and BLIP can be improved and that there are a large number of sequences consistent with tight binding between BLIP and beta-lactamase.     

A broad-spectrum peptide inhibitor of beta-lactamase identified using phage display and peptide arrays

Hydrolysis of beta-lactam antibiotics by beta-lactamase enzymes is the most common mechanism of bacterial resistance to these agents. Several small-molecule, mechanism-based inhibitors of beta-lactamases such as clavulanic acid are clinically available although resistance to these inhibitors has been increasing in bacterial populations. In addition, these inhibitors act only on class A beta-lactamases. Here we utilized phage display to identify peptides that bind to the class A beta-lactamase, TEM-1. The binding affinity of one of these peptides was further optimized by the synthesis of peptide arrays using SPOT synthesis technology. After two rounds of optimization, a linear 6-mer peptide with the sequence RRGHYY was obtained. A soluble version of this peptide was synthesized and found to inhibit TEM-1 beta-lactamase with a K(i) of 136 micro M. Surprisingly, the peptide inhibits the class A Bacillus anthracis Bla1 beta-lactamase with a K(i) of 42 micro M and the class C beta-lactamase, P99, with a K(i) of 140 micro M, despite the fact that it was not optimized to bind these enzymes. This peptide may be a useful starting point for the design of non-beta-lactam, broad-spectrum peptidomimetic inhibitors of beta-lactamases.     

Susceptibility of beta-lactamase to core amino acid substitutions.

To determine which amino acids in TEM-1 beta-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme. A comprehensive screening of these libraries identified several TEM-1 beta-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity. An examination of positions 66 and 76 in the class A beta-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other beta-lactamases. It is possible that position 76 varies in the gene family because beta-lactamase mutants with non-conservative substitutions at position 76 retain partial function. In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function. By determining the beta-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions. An analysis of the Protein Data Bank files for three class A beta-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.     

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii,Citrobacter murliniae,Citrobacter werkmanii,Escherichia fergusonii and Enterobacter cancerogenus

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.     

Expansion of the zinc metallo-hydrolase family of the beta-lactamase fold

Recently, the zinc metallo-hydrolase family of the beta-lactamase fold has grown quite rapidly, accompanied by the accumulation of sequence and structure data. The variety of the biological functions of the family is higher than expected. In addition, the members often have mosaic structures with additional domains. The family includes class B beta-lactamase, glyoxalase II, arylsulfatase, flavoprotein, cyclase/dehydrase, an mRNA 3'-processing protein, a DNA cross-link repair enzyme, a DNA uptake-related protein, an alkylphosphonate uptake-related protein, CMP-N-acetylneuraminate hydroxylase, the romA gene product, alkylsulfatase, and insecticide hydrolases. In this minireview, the functional and structural varieties of the growing protein family are described.     

Irreversible inhibition of the Mycobacterium tuberculosis beta-lactamase by clavulanate

Members of the beta-lactam class of antibiotics, which inhibit the bacterial d,d-transpeptidases involved in cell wall biosynthesis, have never been used systematically in the treatment of Mycobacterium tuberculosis infections because of this organism's resistance to beta-lactams. The critical resistance factor is the constitutive production of a chromosomally encoded, Ambler class A beta-lactamase, BlaC in M. tuberculosis. We show that BlaC is an extended spectrum beta-lactamase (ESBL) with high levels of penicillinase and cephalosporinase activity as well as measurable activity with carbapenems, including imipenem and meropenem. We have characterized the enzyme's inhibition by three FDA-approved beta-lactamase inhibitors: sulbactam, tazobactam, and clavulanate. Sulbactam inhibits the enzyme competitively and reversibly with respect to nitrocefin. Tazobactam inhibits the enzyme in a time-dependent manner, but the activity of the enzyme reappears due to the slow hydrolysis of the covalently acylated enzyme. In contrast, clavulanate reacts with the enzyme quickly to form hydrolytically stable, inactive forms of the enzyme that have been characterized by mass spectrometry. Clavulanate has potential to be used in combination with approved beta-lactam antibiotics to treat multi-drug resistant (MDR) and extremely drug resistant (XDR) strains of M. tuberculosis.     

Kinetics and mechanism of the serine beta-lactamase catalyzed hydrolysis of depsipeptides

Steady-state kinetic parameters have been determined for the hydrolysis of a series of acyclic depsipeptides (ester analogues of acyl-D-alanyl-D-alanine peptides) catalyzed by representative class C (Enterobacter cloacae P99) and class A (Bacillus cereus I, TEM-2, and Staphylococcus aureus PC1) beta-lactamases. The best of these substrates, and the one most used in this work, was m-[[(phenylacetyl)-glycyl]oxy]benzoic acid, whose rates of cleavage could be followed spectrophotometrically. The P99 enzyme also catalyzed the methanolysis of these substrates in aqueous methanol solutions. Quantitative evaluation of the effects of methanol on the kinetics of the competing hydrolysis and methanolysis reactions, and on the product distribution, supports a reaction mechanism involving an acyl-enzyme intermediate whose formation is rate-determining under conditions of substrate saturation. Consideration of the variation of these kinetic parameters with the structure of the depsipeptides and comparison with the analogous parameters for bicyclic beta-lactam substrates suggest that a variety of substrate binding modes exist on this enzyme. The class A enzymes, B. cereus beta-lactamase I and the TEM-2 beta-lactamase, catalyze depsipeptide and benzylpenicillin hydrolyses but not methanolysis. The acyl-enzyme derived from both types of substrate is thus shielded from external nucleophiles; the shielding is therefore not an effect, direct or indirect, of the thiazolidinyl group in the penicilloyl-enzyme. The class A beta-lactamase of the PC1 plasmid of S. aureus is distinctly different from the above two representatives of that class, in that it does catalyze methanolysis of depsipeptides (but not of benzylpenicillin). The methanolysis kinetics suggest that deacylation is rate-determining at saturation, a conclusion supported by the demonstration of an intermediate during the hydrolysis of m-[[(phenylacetyl)glycyl]oxy]benzoate, subsequent to leaving-group departure. The beta-lactamases have thus been shown to catalyze the hydrolysis of specific depsipeptides with comparable facility to that demonstrated by D-alanyl-D-alanine carboxypeptidase/transpeptidases. The former enzymes, however, differ in being unable to cleave the analogous peptides.     

Dissecting the protein-protein interface between beta-lactamase inhibitory protein and class A beta-lactamases

beta-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A beta-lactamases at a wide range of affinities. Alanine-scanning mutagenesis was previously performed to identify the amino acid sequence requirements of BLIP for inhibiting TEM-1 beta-lactamase and SME-1 beta-lactamase. Two hotspots of binding energy, one from each domain of BLIP, were identified (Zhang, Z., and Palzkill, T. (2003) J. Biol. Chem. 278, 45706-45712). This study has been extended to examine the amino acid sequence requirements of BLIP for binding to the SHV-1 beta-lactamase, which is a poor binding substrate (Ki= 1.1 microm), and the Bacillus anthracis Bla1 enzyme (Ki= 2.5 nm). The two hotspots previously identified as important for binding TEM-1 and SME-1 beta-lactamase were also found to be important for binding Bla1. The hotspot from the second domain of BLIP, however, does not make substantial contributions to SHV-1 binding. This may explain why BLIP binds to SHV-1 beta-lactamase with much weaker affinity than to the other three enzymes. Three regions, including two loops that insert into the active pocket of TEM-1 beta-lactamase and the Glu-73-Lys-74 buried charge motif, exhibit strikingly different effects on the binding affinity of BLIP toward the various enzymes when mutated and, therefore, act as specificity determinants. Analysis of double mutants of BLIP that combine specificity-determining residues suggests that these residues contribute to the poor affinity between the second domain of BLIP and SHV-1 beta-lactamase.