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Inactivation of TEM-1 beta-lactamase by 6-acetylmethylenepenicillanic acid.

ABSTRACT: 6-Acetylmethylenepenicillanic acid is a new kinetically irreversible inhibitor of various beta-lactamases. Interaction between 6-acetylmethylenepenicillanate and purified TEM-1 beta-lactamase during the inactivation process was investigated. 6-Acetylmethylenepenicillanate inhibited the enzyme in a second-order fashion with a rate constant of 0.61 microM-1 . S-1. The apparent inactivation constant decreased in the presence of increasing concentrations of the substrate benzylpenicillin. Native enzyme (pI 5.4) was converted into two inactive forms with pI 5.25 and 5.15, the latter form being transient and readily converted into the more stable form with pI 5.15. Even a 50-fold excess of inhibitor over enzyme did not produce any other inactivated species of the enzyme. All the results obtained suggest that 6-acetylmethylenepenicillanate is a potent irreversible and active-site-directed inhibitor of TEM-1 beta-lactamase.

SUBMITTER: Arisawa M

PROVIDER: S-EPMC1154137 | biostudies-other | 1983 Mar

REPOSITORIES: biostudies-other

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Mechanism of inactivation of TEM-1 beta-lactamase by 6-acetylmethylenepenicillanic acid.

Project description:The interaction between 6-acetylmethylenepenicillanic acid (compound Ro 15-1903; AMPA) and TEM-1 beta-lactamase was investigated in order to elucidate the mechanism of action of AMPA. Formation of the enzyme-inhibitor complex (EA) was accompanied by a shift of the absorbance maximum from 292 nm to 303 nm and an increase in the absorption. Regeneration of activity was very slow and incomplete, reaching about one-third of the initial activity after 48 h at 37 degrees C. This behaviour indicated a branched pathway of the decay of the inactivated enzyme. Kinetic and isoelectric-focusing experiments proved this assumption. The first-order constant of regeneration of active enzyme was 6 X 10(-6)-10 X 10(-6) s-1, whereas the rate constant leading to inactive enzyme (EA') was 10 X 10(-6)-15 X 10(-6) s-1 at pH 7.0. Both constants became larger at higher pH. Inactive enzyme (EA') consisted of two major species, with pI 5.36 (EA'1) and 5.30 (EA'2). The former increased at the beginning of incubation but decreased after prolonged incubation. From consideration of these results and previous data [Arisawa & Then (1983) Biochem. J. 209, 609-615], a likely mechanism of inactivation of TEM-1 beta-lactamase by AMPA is discussed.

| S-EPMC1154378 | biostudies-other

TEM-109 (CMT-5), a natural complex mutant of TEM-1 beta-lactamase combining the amino acid substitutions of TEM-6 and TEM-33 (IRT-5).

Project description:Escherichia coli CF349 exhibited a complex beta-lactam resistance phenotype, including resistance to amoxicillin and ticarcillin alone and in combination with clavulanate and to some extended-spectrum cephalosporins. The double-disk synergy test was positive. CF349 harbored an 85-kb conjugative plasmid which encoded a beta-lactamase of pI 5.9. The corresponding bla gene was identified by PCR and sequencing as a bla(TEM) gene. The deduced protein sequence revealed a new complex mutant of TEM-1 beta-lactamase designated TEM-109 (CMT-5). TEM-109 contained both the substitutions Glu104Lys and Arg164His of the expanded-spectrum beta-lactamase (ESBL) TEM-6 and Met69Leu of the inhibitor-resistant TEM-33 (IRT-5). TEM-109 exhibited hydrolytic activity against ceftazidime similar to that of TEM-6 (k(cat), 56 s(-1) and 105 s(-1), respectively; K(m) values, 226 and 247 microM, respectively). The 50% inhibitory concentrations of clavulanate and tazobactam (0.13 microM and 0.27 microM, respectively) were 5- to 10-fold higher for TEM-109 than for TEM-6 (0.01 and 0.06 microM, respectively) but were almost 10-fold lower than those for TEM-33. The characterization of this novel CMT, which exhibits a low level of resistance to inhibitors, highlights the emergence of this new ESBL type.

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Fitness Effects of Single Amino Acid Insertions and Deletions in TEM-1 β-Lactamase.

Project description:Short insertions and deletions (InDels) are a common type of mutation found in nature and a useful source of variation in protein engineering. InDel events have important consequences in protein evolution, often opening new pathways for adaptation. However, much less is known about the effects of InDels compared to point mutations and amino acid substitutions. In particular, deep mutagenesis studies on the distribution of fitness effects of mutations have focused almost exclusively on amino acid substitutions. Here, we present a near-comprehensive analysis of the fitness effects of single amino acid InDels in TEM-1 β-lactamase. While we found InDels to be largely deleterious, partially overlapping deletion-tolerant and insertion-tolerant regions were observed throughout the protein, especially in unstructured regions and at the end of helices. The signal sequence of TEM-1 tolerated InDels more than the mature protein. Most regions of the protein tolerated insertions more than deletions, but a few regions tolerated deletions more than insertions. We examined the relationship between InDel tolerance and a variety of measures to help understand its origin. These measures included evolutionary variation in β-lactamases, secondary structure identity, tolerance to amino acid substitutions, solvent accessibility, and side-chain weighted contact number. We found secondary structure, weighted contact number, and evolutionary variation in class A beta-lactamases to be the somewhat predictive of InDel fitness effects.

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Updated sequence information for TEM beta-lactamase genes.

Project description:The sequences of the promoter regions and of the structural genes for 13 penicillinase, extended-spectrum, and inhibitor-resistant TEM-type beta-lactamases have been determined, and an updated blaTEM gene nomenclature is proposed.

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CMT-type beta-lactamase TEM-125, an emerging problem for extended-spectrum beta-lactamase detection.

Project description:The clinical strain Escherichia coli TO799 was resistant to penicillin-clavulanate combinations and ceftazidime and was not reproducibly detected as an extended-spectrum beta-lactamase (ESBL) according to the standards of the Clinical Laboratory Standards Institute (CLSI; formerly NCCLS) and the national guidelines of the French Society for Microbiology (Comité de l'Antibiogramme de la Société Française de Microbiologie). A novel beta-lactamase, designated TEM-125, was responsible for this phenotype. TEM-125 harbors a complex association of mutations previously described in the ESBL TEM-12 and in the inhibitor-resistant beta-lactamase TEM-39. TEM-125 is the first complex mutant TEM to present hydrolytic activity against ceftazidime (kcat, 3.7 s(-1)) together with a high level of resistance to clavulanate (50% inhibitory concentration, 13.6 microM). The discovery of such an ESBL, which is difficult to detect by the usual ESBL detection methods, confirms the emergence of a complex mutant TEM subgroup and highlights the need to evaluate detection methods so as to avoid possible therapeutic failures.

| S-EPMC1489774 | biostudies-literature

TEM-121, a novel complex mutant of TEM-type beta-lactamase from Enterobacter aerogenes.

Project description:Enterobacter aerogenes clinical isolate LOR was resistant to penicillins and ceftazidime but susceptible to cefuroxime, cephalothin, cefoxitin, cefotaxime, ceftriaxone, and cefepime. PCR and cloning experiments from this strain identified a novel TEM-type beta-lactamase (TEM-121) differing by five amino acid substitutions from beta-lactamase TEM-2 (Glu104Lys, Arg164Ser, Ala237Thr, Glu240Lys, and Arg244Ser) and by only one amino acid change from the extended-spectrum beta-lactamase (ESBL) TEM-24 (Arg244Ser), with the last substitution also being identified in the inhibitor-resistant beta-lactamase IRT-2. Kinetic parameters indicated that TEM-121 hydrolyzed ceftazidime and aztreonam (like TEM-24) and was inhibited weakly by clavulanic acid and strongly by tazobactam. Thus, TEM-121 is a novel complex mutant TEM beta-lactamase (CMT-4) combining the kinetic properties of an ESBL and an inhibitor-resistant TEM enzyme.

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A novel extended-spectrum TEM-type beta-lactamase, TEM-138, from Salmonella enterica serovar Infantis.

Project description:A novel natural TEM beta-lactamase with extended-spectrum activity, TEM-138, was identified in a ceftazidime-resistant clinical isolate of Salmonella enterica serovar Infantis. Compared to TEM-1, TEM-138 contains the following mutations: E104K, N175I, and G238S. The bla(TEM-138) gene was located on a 50-kb transferable plasmid. Expression studies with Escherichia coli revealed efficient ceftazidimase and cefotaximase activities for TEM-138.

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Novel TEM-type extended-spectrum beta-lactamase, TEM-134, in a Citrobacter koseri clinical isolate.

Project description:A new natural TEM derivative with extended-spectrum beta-lactamase activity, TEM-134, was identified in a ceftazidime-resistant clinical isolate of Citrobacter koseri. Compared to TEM-1, TEM-134 contains the following mutations: Q39K, E104K, R164H, and G238S. The bla(TEM-134) gene was not transferable by conjugation and, apparently, was chromosomally encoded. Expression studies with Escherichia coli revealed efficient cefotaximase and ceftazidimase activity for TEM-134.

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Kinetics of inactivation of beta-lactamase I by 6 beta-bromopenicillanic acid.

Project description:The kinetics of the inactivation of beta-lactamase I from Bacillus cereus 569 by preparations of 6 alpha-bromopenicillanic acid showed unexpected features. These can be quantitatively accounted for on the basis of the inactivator being the epimer, 6 beta-bromopenicillanic acid. At pH 9.2, the rate-determining step in the inactivation is the formation of the inactivator. When pure 6 beta-bromopenicillanic acid is used to inactivate beta-lactamase I, simple second-order kinetics are observed. The inactivated enzyme has a new absorption peak at 326 nm. The rate constant for inactivation has the same value as the rate constant for appearance of absorption at 326 nm; the rate-determining step may thus be fission of the beta-lactam ring of 6 beta-bromopenicillanic acid. Inactivation is slower in the presence of substrate, and the observed kinetics can be quantitatively accounted for on a simple competitive model. The results strongly suggest that inactivation is a consequence of reaction at the active site.

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