Inhibition of TEM-2 beta-lactamase from Escherichia coli by clavulanic acid: observation of intermediates by electrospray ionization mass spectrometry

Clavulanic acid, the therapeutically important inhibitor of beta-lactamases containing a nucleophilic serine residue at their active sites, inhibits Escherichia coli TEM-2 beta-lactamase via a complex mechanism. Electrospray ionization mass spectrometry (ESIMS) studies revealed that a minimum of four different modified proteins are formed upon incubation of clavulanate with the TEM-2 enzyme. These exhibit mass increments relative to the unmodified TEM-2 beta-lactamase of 52, 70, 88, and 155 Da. Time course studies implied that no long-lived forms of clavulanate-inhibited TEM-2 beta-lactamase retain the carbons of the oxazolidine ring of clavulanate. The absence of a 199 Da increment to unmodified TEM-2 suggests rapid decarboxylation of clavulanate upon binding to the enzyme. Proteolytic digestions of purified forms of clavulanate inhibited TEM-2 beta-lactamase followed by analyses using high-performance liquid chromatography coupled to ESIMS (HPLC-ESIMS) and chemical sequencing were used to provide positional information on the modifications to the enzyme. Increments of 70 and 80 Da increments were shown to be located in a peptide containing Ser-70. A further 70 Da mass increment, assigned as a beta-linked acrylate, was localized to a peptide containing Ser-130. A mechanistic scheme for the reaction of clavulanate with TEM-2 beta-lactamase is proposed in which acylation at Ser-70 and subsequent decarboxylation is followed either by cross-linking with Ser-130 to form a vinyl ether or by reformation of unmodified enzyme via a Ser-70 linked (hydrated) aldehyde. Purified cross-linked vinyl ether was observed to slowly convert under acidic conditions to a Ser-70 linked (hydrated) aldehyde with concomitant conversion of Ser-130 to a dehydroalanyl residue.     

Reversal of clavulanate resistance conferred by a Ser-244 mutant of TEM-1 beta-lactamase as a result of a second mutation (Arg to Ser at position 164) that enhances activity against ceftazidime

The mutation of Arg-244 to Ser (Arg-244-->Ser mutation) in the TEM-1 beta-lactamase has been shown to produce resistance to inactivation by clavulanate in the mutant enzyme and resistance to ampicillin plus clavulanate in a strain of Escherichia coli producing this enzyme. The Arg-164-->Ser mutation in the TEM-1 beta-lactamase (TEM-12 enzyme) is known to enhance the activity of the enzyme against ceftazidime, resulting in resistance to the drug in a strain producing the mutant enzyme (D. A. Weber, C. C. Sanders, J. S. Bakken, and J. P. Quinn, J. Infect. Dis. 162:460-465, 1990). The doubly mutated derivative of the TEM-1 enzyme (Ser-164/Ser-244) retains the characteristics of the Ser-164 mutant enzyme, i.e., enhanced activity against ceftazidime and sensitivity to inactivation by clavulanate. It also confers the same phenotype as the Ser-164 mutant enzyme, i.e., resistance to ceftazidime and ampicillin, with reversal of this resistance in the presence of clavulanate. Thus, the Arg-164-->Ser mutation in the TEM-1 beta-lactamase suppresses the effect of the Arg-244-->Ser mutation which, by itself, reduces the sensitivity of the enzyme to inactivation by clavulanate.     

A TEM-derived extended-spectrum beta-lactamase in Pseudomonas aeruginosa

A clinical strain of Pseudomonas aeruginosa, PAe1100, was found to be resistant to all antipseudomonal beta-lactam antibiotics and to aminoglycosides, including gentamicin, amikacin, and isepamicin. PAe1100 produced two beta-lactamases, TEM-2 (pI 5.6) and a novel, TEM-derived extended-spectrum beta-lactamase called TEM-42 (pI 5.8), susceptible to inhibition by clavulanate, sulbactam, and tazobactam. Both enzymes, as well as the aminoglycoside resistance which resulted from AAC(3)-IIa and AAC(6')-I production, were encoded by an 18-kb nonconjugative plasmid, pLRM1, that could be transferred to Escherichia coli by transformation. The gene coding for TEM-42 had four mutations that led to as many amino acid substitutions with respect to TEM-2: Val for Ala at position 42 (Ala42), Ser for Gly238, Lys for Glu240, and Met for Thr265 (Ambler numbering). The double mutation Ser for Gly238 and Lys for Glu240, which has so far only been described in SHV-type but not TEM-type enzymes, conferred concomitant high-level resistance to cefotaxime and ceftazidime. The novel, TEM-derived extended-spectrum beta-lactamase appears to be the first of its class to be described in P. aeruginosa.     

Cloning and sequencing of the gene encoding Toho-2, a class A beta-lactamase preferentially inhibited by tazobactam

Escherichia coli TUM1083, which is resistant to ampicillin, carbenicillin, cephaloridine, cephalothin, piperacillin, cefuzonam, and aztreonam while being sensitive to cefoxitin, moxalactam, cefmetazole, ceftazidime, and imipenem, was isolated from the urine of a patient treated with beta-lactam antibiotics. The beta-lactamase (Toho-2) purified from the bacteria hydrolyzed beta-lactam antibiotics such as penicillin G, carbenicillin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam and especially had increased relative hydrolysis rates for cephalothin, cephaloridine, cefotaxime, and ceftizoxime. Different from other extended-spectrum beta-lactamases, Toho-2 was inhibited 16-fold better by the beta-lactamase inhibitor tazobactam than by clavulanic acid. Resistance to beta-lactams was transferred by conjugation from E. coli TUM1083 to E. coli ML4909, and the transferred plasmid was about 54.4 kbp, belonging to the incompatibility group IncFII. The cefotaxime resistance gene for Toho-2 was subcloned from the 54.4-kbp plasmid. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 981 bases. The nucleotide sequence of the gene (DDBJ accession no. D89862) designated as bla(toho) was found to have 76.3% identity to class A beta-lactamase CTX-M-2 and 76.2% identity to Toho-1. It has 55.9% identity to SHV-1 beta-lactamase and 47.5% identity to TEM-1 beta-lactamase. Therefore, the newly isolated beta-lactamase designated as Toho-2 produced by E. coli TUM1083 is categorized as an enzyme similar to Toho-1 group beta-lactamases rather than to mutants of TEM or SHV enzymes. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 327 amino acid residues. Comparison of Toho-2 with other beta-lactamase (non-Toho-1 group) suggests that the substitutions of threonine for Arg-244 and arginine for Asn-276 are important for the extension of the substrate specificity.     

Transcontinental importation into the UK of Escherichia coli expressing a plasmid-mediated AmpC-type beta-lactamase exposed during an outbreak of SHV-5 extended-spectrum beta-lactamase in a Leeds hospital

Sixteen strains of Escherichia coli with high-level resistance to extended-spectrum cephalosporins and other classes of antibiotic have been isolated at St James' University Hospital, Leeds. They produce up to three separate beta-lactamases: TEM-1, SHV-5 and, in five isolates, a plasmid-mediated AmpC-type enzyme. With the exception of carbapenems, the isolates reported in this study were resistant to all beta-lactam antibiotics including extended-spectrum cephalosporins and the monobactam aztreonam. There was evidence of the spread of a plasmid encoding SHV-5, particularly amongst patients on the liver transplant unit. Sensitivity to beta-lactam antibiotics in five isolates expressing the AmpC-type beta-lactamase was not restored by the beta-lactamase inhibitor clavulanic acid. These bacteria also carried blaSHV-5 on a large plasmid. PCR-amplification of the structural gene and digestion with restriction endonucleases demonstrated that the plasmid-mediated blaAmpC probably identified as BIL-1 using the criteria available. Four of the five patients carrying isolates that carried the plasmid-located blaAmpC gene had recently visited the Indian subcontinent and we presume that they returned carrying these bacteria. Restriction fragment length polymorphism analysis using pulsed field gel electrophoresis (PFGE) suggests that at least four distinct strains existed amongst these five isolates. The two isolates that had very similar PFGE patterns had different plasmid profiles and were isolated from different locations in the hospital and at different times. This study demonstrates the ease with which highly resistant bacteria can be imported into the UK and spread within hospitals.     

Ceftazidime-resistant Klebsiella pneumoniae and Escherichia coli isolates producing TEM-10 and TEM-43 beta-lactamases from St. Louis, Missouri

Ceftazidime-resistant Escherichia coli and Klebsiella pneumoniae (49 and 102 isolates, respectively) were collected from Barnes-Jewish Hospital, St. Louis, Mo., from 1992 to 1996. They were uniformly resistant to ceftazidime, generally resistant to aztreonam, and variably susceptible to cefotaxime. Four representative E. coli strains and 15 Klebsiella strains were examined. From one to four beta-lactamases were produced per strain, with three possible enzymes related to ceftazidime resistance: enzymes with pI values of 5.6, 6.1, or 7.6. By pulsed-field gel electrophoresis there were at least 13 different Klebsiella strain types and 3 different E. coli strain types, indicating that the outbreak was not clonal. After cloning and sequencing of the beta-lactamase-encoding genes, the enzyme with a pI of 5.6 was identified as TEM-10. The enzyme with a pI of 6.1 was a novel TEM variant (TEM-43) with Lys at 104, His at 164, and Thr at 182. TEM-43 showed broad-spectrum hydrolytic activity against all penicillins, with the highest hydrolysis rate for ceftazidime compared to those for the other expanded-spectrum cephalosporins. Aztreonam was also a good substrate for TEM-43, with hydrolytic activity similar to that of ceftazidime and affinity higher than that of ceftazidime. The TEM-43 beta-lactamase was well inhibited by clavulanic acid and tazobactam at concentrations of < 10 nM. Sulbactam was less effective than the other inhibitors. The Thr182 mutation previously reported in an inhibitor-resistant beta-lactamase did not cause the TEM-43 enzyme to become resistant to any of the inhibitors.     

Selection and characterization of beta-lactam-beta-lactamase inactivator-resistant mutants following PCR mutagenesis of the TEM-1 beta-lactamase gene

Mechanism-based inactivators of beta-lactamases are used to overcome the resistance of clinical pathogens to beta-lactam antibiotics. This strategy can itself be overcome by mutations of the beta-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 beta-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with beta-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp beta-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM beta-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.     

Selection strategy for site-directed mutagenesis based on altered beta-lactamase specificity

Conventional approaches to oligonucleotide-directed mutagenesis rely upon the application of a selection strategy to maximize mutagenesis efficiencies. We have developed a mutagenesis procedure that incorporates a novel antibiotic resistance for selection. The selection involves altering the substrate specificity of TEM-1 beta-lactamase, the enzyme responsible for bacterial resistance to beta-lactam antibiotics such as ampicillin. The gene encoding beta-lactamase is commonly found on cloning and shuttle vectors used in molecular biology. Amino acid substitutions in several active site residues of beta-lactamase result in increased hydrolytic activity against extended-spectrum penicillins and cephalosporins. This increased activity confers a novel resistance specific to the mutant and thus provides the basis of the selection strategy. We describe a simple and efficient mutagenesis procedure and its application to creating a range of oligonucleotide-directed mutants.     

Glycohemoglobin filter assay for doctors' offices based on boronic acid affinity principle

We present a new filter assay for the determination of glycohemoglobin as a unique application of the boronic acid affinity principle. With the use of a water-soluble blue-colored boronic acid derivative and a specific precipitation method for hemoglobin, total hemoglobin including bound boronic acid is precipitated and collected on a filter strip before quantification. Hemoglobin and boronic acid are quantified by a dual-wave-length reflectometric measurement, and the result is reported directly as percent glycohemoglobin. The test is simple, quick, and designed as a doctors' office test for the monitoring and management of diabetes. The imprecision of the assay is < 4% over the range 3-18% Hb A1c, and the method is linear up to at least 20% Hb A1c. Comparisons with four well-established glycohemoglobin methods yielded correlation coefficients ranging from 0.94 to 0.99, with slopes from 0.94 to 1.01.     

The role of residue 238 of TEM-1 beta-lactamase in the hydrolysis of extended-spectrum antibiotics

beta-Lactamases inactivate beta-lactam antibiotics by catalyzing the hydrolysis of the amide bond in the beta-lactam ring. The plasmid-encoded class A TEM-1 beta-lactamase is a commonly encountered beta-lactamase. It is able to inactivate penicillins and cephalosporins but not extended-spectrum antibiotics. However, TEM-1-derived natural variants containing the G238S amino acid substitution display increased hydrolysis of extended-spectrum antibiotics. Two models have been proposed to explain the role of the G238S substitution in hydrolysis of extended-spectrum antibiotics. The first proposes a direct hydrogen bond of the Ser238 side chain to the oxime group of extended-spectrum antibiotics. The second proposes that steric conflict with surrounding residues, due to increased side chain volume, leads to a more accessible active site pocket. To assess the validity of each model, TEM-1 mutants with amino acids substitutions of Ala, Ser, Cys, Thr, Asn, and Val have been constructed. Kinetic analysis of these enzymes with penicillins and cephalosporins suggests that a hydrogen bond is necessary but not sufficient to achieve the hydrolytic activity of the G238S enzyme for the extended-spectrum antibiotics cefotaxime and ceftazidime. In addition, it appears that the new hydrogen bond interaction is to a site on the enzyme rather than directly to the extended-spectrum antibiotic. The data indicate that, for the G238S substitution, a combination of an optimal side chain volume and hydrogen bonding potential results in the most versatile and advantageous antibiotic hydrolytic spectrum for bacterial resistance to extended-spectrum antibiotics.     

Characterization of ceftriaxone-resistant Enterobacteriaceae: a multicentre study in 26 French hospitals. Vigil'Roc Study Group

During a multicentre study performed in 26 French hospitals, 287 (3.2%) of 9038 Enterobacteriaceae isolated, mainly Enterobacter spp., Serratia spp., Citrobacter spp. and Klebsiella spp. were classified as ceftriaxone resistant on the basis of an MIC > 4 mg/L or the presence of an extended-spectrum beta-lactamase. Extended-spectrum beta-lactamase was present mainly in Klebsiella pneumoniae (65 strains, 10.2%) and very rarely in Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, Citrobacter spp. and Enterobacter spp. The extended-spectrum beta-lactamases conferred low-level resistance to ceftriaxone in nearly 60% of the strains harbouring them, emphasizing the need for routine testing for the presence of these enzymes. Among transconjugants three types of extended-spectrum beta-lactamase were identified. Those resembling TEM-3 were the most common, but TEM-21, and SHV-4 were also found. Clavulanate and to a lesser extent sulbactam inhibited all the extended-spectrum beta-lactamases encountered in this study.     

Cloning and nucleotide sequence analysis of a gene encoding an OXA-derived beta-lactamase in Acinetobacter baumannii

A clinical strain of Acinetobacter baumannii (strain Ab41) that was resistant to all beta-lactam antibiotics tested except ceftazidime, ceftriaxone, ceftizoxime, and imipenem produced three beta-lactamases: a presumptive chromosomal cephalosporinase, a TEM-1-like beta-lactamase (pI 5.4), and a novel OXA-derived beta-lactamase named OXA-21 (pI 7.0). The gene encoding OXA-21 was located in an integron. The nucleotide sequence showed three mutations compared with the sequence of OXA-3, with two being silent; the nonsilent mutation generated a substitution of Ile-217 to Met.     

The effect of high-frequency random mutagenesis on in vitro protein evolution: a study on TEM-1 beta-lactamase

For a number of years a major limitation in genetic analysis of protein function has been the inability to introduce multiple substitutions at distant sites that would enable the selection of clusters of mutations required for improved or novel biological functions. In order to achieve this, we have recently developed a novel mutagenesis procedure in which the triphosphate derivatives of a pyrimidine (6-(2-deoxy-beta-d-ribofuranosyl)-3, 4-dihydro-8H-pyrimido-[4,5-c][1,2]oxazin-7-one; dP) and a purine (8-oxo-2'-deoxyguanosine; 8-oxodG) nucleoside analogue are employed in DNA synthesis reactions in vitro. The procedure allows control of the mutational load and can yield frequencies of amino acid residue substitutions at least one order of magnitude greater than those previously achieved. Here we report the results of an experiment in which we have hypermutated the bacterial enzyme TEM-1 beta-lactamase and selected small pools (<1.5x10(5)) of clones for enzymatic activity against the beta-lactam antibiotic cefotaxime. The experiment resulted in the isolation of a number of TEM-1 mutants with greatly improved activity against cefotaxime. Among these, clone 3D.5 (E104K:M182T:G238S) exhibited a minimum inhibitory concentration for cefotaxime 20,000-fold higher than wild-type TEM-1 and a catalytic efficiency (kcat/Km) 2383 times higher than the wild-type enzyme. Thus, small pools of hypermutated sequences enabled the selection of one of the most active extended beta-lactamases described so far. These results argue against the accepted view that multiple rounds of low-rate mutagenesis and stepwise selection are essential for in vitro protein evolution and extend the scope of directed molecular evolution to proteins for which no genetic selection is available.     

Perspectives for the development of vaccines and immunotherapy against cervico-uterine cancer]

The kinetic parameters of three IRT (Inhibitor-Resistant-TEM-derived-) beta-lactamases (IRT-5, IRT-6 and IRT-I69) were determined for substrates and the beta-lactamase inhibitors: clavulanic acid, sulbactam and tazobactam, and compared with those of TEM-1 beta-lactamase. The catalytic behaviour of the beta-lactamases towards substrates and inhibitors was correlated with the properties of the amino acid at position ABL69. The three IRT beta-lactamases contain at that position a residue Ile, Leu and Val, amino acids whose side-chain are branched. Molecular modelling shows that the methyl groups of Ile-69 (C gamma 2) and Val-69 (C gamma 1) produced steric constraints with the side chain of Asn-170 as well as the main chain nitrogen of Ser-70, a residue contributing to the oxyanion hole. We suggest that hydrophobicity could be the main factor responsible for the kinetic properties of Met69Leu (IRT-5), as no steric effects could be detected by molecular modelling. Hydrophobicity and steric constraints are combined in Met69Ile and Met69Val, IRT-I69 and IRT-6, respectively.     

Cascade testing for the identification of carriers of cystic fibrosis

Purified preparations of TEM-2, P99, Bacillus cereus I and B. cereus II beta-lactamases were examined by electrospray (ES) mass spectrometry. The ES mass spectra of the B. cereus enzymes revealed the presence of four to five components of different mass, corresponding to the loss of different numbers of N-terminal amino acids (ragged ends). The ES mass spectra of both TEM-2 and P99 consisted of a single component with no evidence of ragged ends. All four beta-lactamase preparations were visualized on isoelectric focusing (IEF) gels stained with nitrocefin to investigate a possible correlation between IEF patterns and ragged ends. Multiple banding patterns were seen with each beta-lactamase preparation. Although these may correlate with the presence of ragged ends in the two B. cereus preparations, the satellite bands seen with P99 and TEM-2 were not associated with differences detected by ES mass spectrometry. In this study we have shown for the first time that beta-lactamase satellite bands seen on IEF are not always associated with ragged ends. Furthermore, we have illustrated the use of ES mass spectrometry to characterize the extent of ragged end formation in protein samples. This is of particular significance if the sample is required for detailed biochemical or crystallography experiments.     

A237T as a modulating mutation in naturally occurring extended-spectrum TEM-type beta-lactamases

A TEM-1 beta-lactamase derivative containing the single amino acid substitution A237T slightly increased (from 24 to 32 microg/ml) the cephalothin MIC for Escherichia coli RYC1000 but did not influence the activities of cefotaxime, ceftazidime, and aztreonam (MICs of 0.03, 0.12, and 0.06 microg/ml, respectively). Despite its apparent neutrality, addition of the A237T mutation to the pair of mutations characterizing TEM-10 (R164S and E240K) had a strong effect on substrate preference. Ceftazidime and aztreonam MICs decreased from 128 and 16 microg/ml to 16 and 2 microg/ml, respectively. In contrast, the cefotaxime MIC increased from 0.5 to 4 microg/ml. The acquisition of apparently neutral or even deleterious mutations results in a very effective mechanism of resistance to different beta-lactams that may be simultaneously or subsequently present in the environment. We propose here that the mutation in position 237 is an example of a modulating mutation and that consideration of this type of mutation may be important for understanding the evolution of beta-lactamases.     

Characterization of plasmids which mediate resistance to multiple antibiotics in gram-negative bacteria of nosocomial origin

BACKGROUND: The genetic and molecular mechanisms involved in antimicrobial resistance of 10 strains of gramnegative bacilli (1 Serratia marcescens; 2 Escherichia coli; 1 Proteus mirabilis; 4 Klebsiella pneumoniae; 1 Enterobacter cloacae y 1 Alcaligenes faecalis), isolated from adult patients with nosocomial pulmonary infection at the in-patient facilities of the University Hospital of Los Andes, Mérida, Venezuela, have been studied. METHODS: The antimicrobial susceptibility was determined by minimum inhibitory concentrations using the dilution method in agar. The study of extrachromosomal genes was carried out by conjugation, bacterial infection with the bacteriophage M13 and curing of plasmid by acridine orange. The plasmids were isolated by alkaline lysis and analysis of restriction endonuclease digestion was carried out separately using the enzymes EcoRI and HindIII. A DNA probe, derived from the region which encodes the TEM-1 beta-lactamase of the plasmid pBR322 was used for dot-blot hybridization tests. RESULTS: All of the gramnegative bacilli showed resistance to ampicillin, carbenicillin and cephalothin (> 128 micrograms/ml) and 3 strains also showed resistance to gentamicin (> 64 micrograms/ml). Genetic and molecular procedures showed the presence of conjugative plasmids of approximately 54 kb in all the 10 strains. The restriction patterns obtained by using EcoRI and HindIII indicated common DNA fragments in most of the plasmids studied. The dot-blot hybridization tests confirmed homology between the plasmids and the DNA probe used (TEM-1 beta-lactamase). CONCLUSIONS: In this study, the gramnegative bacteria of nosocomial origin harbored self-transferable plasmids of approximately 54 kb, which mediate resistance to gentamicin and encode a beta-lactamase of the TEM group.     

Display of functional beta-lactamase inhibitory protein on the surface of M13 bacteriophage

The display of proteins on the surface of filamentous phage has been shown to be a powerful method to select variants of a protein with altered binding properties from large combinatorial libraries of mutants. The beta-lactamase inhibitory protein (BLIP) is a 165-amino-acid protein that binds and inhibits TEM-1 beta-lactamase-catalyzed hydrolysis of the penicillin and cephalosporin antibiotics. Here we describe the construction of a new phagemid vector and the use of this vector to display BLIP on the surface of filamentous phage. It is shown that BLIP-displaying phage bind to immobilized beta-lactamase and that the binding can be competed off by the addition of soluble beta-lactamase. In addition, a two-step phage enzyme-linked immunosorbent assay procedure was used to demonstrate that the BLIP-displaying phage bind beta-lactamase with a 50% inhibitory concentration of 1 nM, which compares favorably with a previously published Ki of 0.6 nM. A system has therefore been established for protein engineering of BLIP to expand its range of binding to other beta-lactamases and penicillin-binding proteins.     

Characterization of a PSE-4 mutant with different properties in relation to penicillanic acid sulfones: importance of residues 216 to 218 in class A beta-lactamases

Class A beta-lactamases are inactivated by the suicide inactivators sulbactam, clavulanic acid, and tazobactam. An examination of multiple alignments indicated that amino acids 216 to 218 differed among class A enzymes. By random replacement mutagenesis of codons 216 to 218 in PSE-4, a complete library consisting of 40,864 mutants was created. The library of mutants with mutations at positions 216 to 218 in PSE-4 was screened on carbenicillin and ampicillin with the inactivator sulbactam; a collection of 14 mutants was selected, and their bla genes were completely sequenced. Purified wild-type and mutant PSE-4 beta-lactamases were used to measure kinetic parameters. One enzyme, V216S:T217A:G218R, was examined for its peculiar pattern of inhibition. There was an increase in the Km from 68 microM for the wild type to 271 microM for the mutant for carbenicillin and 33 to 216 microM for ampicillin. Relative to the wild-type PSE-4 enzyme, 37- and 30-fold increases in Ki values were observed for the mutant enzyme for sulbactam and tazobactam, respectively. The results that were obtained suggested that positions 216 to 218 are important for interactions with penicillanic acid sulfone inhibitors. In contrast, V216 and A217 in the TEM-1 class A beta-lactamase do not tolerate amino acid residue substitutions. However, for the PSE-4 beta-lactamase, 11 of 14 mutants from the library of mutants with mutations at positions 216 to 218 whose sequences were determined had substitutions at position 216 (G, R, A, S) and position 217 (A, S). The data showed the importance of residues 216 to 218 in their atomic interactions with inactivators in the PSE-4 beta-lactamase structure.     

Inactivation of beta-lactamase from Shigella flexneri UCSF-129 by BRL 42715, a potent inhibitor

The effect of a powerful inhibitor, BRL 42715, on beta-lactamase from Shigella flexneri UCSF-129, to overcome the problem of shigellosis and its resistance to ampicillin, was studied. The I50 was determined for BRL 42715 [C6-(N1-methyl-1,2,3 triazolylmethylene)penem] as 0.0049 microgram/ml being 20-fold lower than the best inhibitor, 6-beta-iodopenicillanic acid, previously reported. The MIC fell from 2,048 to 2 micrograms/ml in the presence of 1 microgram/ml of BRL 42715. The synergism of the ampicillin plus this inhibitor lasted for 9 h. BRL 42715 is an irreversible inhibitor, according to the dialysis results, and a substrate analogue.