Dataset Information

Structural Basis for Enhancement of Carbapenemase Activity in the OXA-51 Family of Class D ?-Lactamases.

ABSTRACT: Class D ?-lactamases of Acinetobacter baumannii are enzymes of the utmost clinical importance, producing resistance to last resort carbapenem antibiotics. Although the OXA-51-like enzymes constitute the largest family of class D ?-lactamases, they are poorly studied and their importance in conferring carbapenem resistance is controversial. We present the detailed microbiological, kinetic, and structural characterization of the eponymous OXA-51 ?-lactamase. Kinetic studies show that OXA-51 has low catalytic efficiency for carbapenems, primarily due to the low affinity of the enzyme for these substrates. Structural studies demonstrate that this low affinity results from the obstruction of the enzyme active site by the side chain of Trp222, which presents a transient steric barrier to an incoming carbapenem substrate. The Trp222Met substitution relieves this steric hindrance and elevates the affinity of the mutant enzyme for carbapenems by 10-fold, significantly increasing the levels of resistance to these antibiotics. The ability of OXA-51 to evolve into a robust carbapenemase as the result of a single amino acid substitution may, in the near future, elevate the ubiquitous enzymes of the OXA-51 family to the status of the most deleterious A. baumannii carbapenemases, with dire clinical consequences.

SUBMITTER: Smith CA

PROVIDER: S-EPMC4546549 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Publications

Structural Basis for Enhancement of Carbapenemase Activity in the OXA-51 Family of Class D β-Lactamases.

Smith Clyde A CA Antunes Nuno Tiago NT Stewart Nichole K NK Frase Hilary H Toth Marta M Kantardjieff Katherine A KA Vakulenko Sergei S

ACS chemical biology 20150612 8

Class D β-lactamases of Acinetobacter baumannii are enzymes of the utmost clinical importance, producing resistance to last resort carbapenem antibiotics. Although the OXA-51-like enzymes constitute the largest family of class D β-lactamases, they are poorly studied and their importance in conferring carbapenem resistance is controversial. We present the detailed microbiological, kinetic, and structural characterization of the eponymous OXA-51 β-lactamase. Kinetic studies show that OXA-51 has lo ...[more]

PMID: 26042471

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Project description:We investigated the mechanisms involved in imipenem resistance in 23 clinical strains of Acinetobacter baumannii. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the presence of a 30-kDa protein in imipenem-intermediate A. baumannii (IIAB) and imipenem-resistant A. baumannii (IRAB) strains; this protein was almost undetectable in imipenem-susceptible A. baumannii (ISAB) strains. The 30-kDa protein was identified as an OXA-51-like carbapenemase using two-dimensional gel electrophoresis and mass spectrometry. Similar to other recent findings, bla(OXA-51-like) genes were found to exist in all 23 clinical strains; however, the transcript levels of bla(OXA-51-like) in the IIAB and IRAB were higher than in the ISAB strains using reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays. This change was due to the presence of an insertion sequence, ISAba1, upstream of bla(OXA-51-like) in the IIAB and IRAB strains that was not present in the ISAB strains. The introduction of bla(OXA-66) (a bla(OXA-51)(-like) gene), identified in ISAB ab1254 and IRAB ab1266, into Escherichia coli TOP10 cells resulted in 3.95-fold and 7.90-fold elevations in resistance to imipenem, respectively. Furthermore, when ISAB ab8 and ISAB ab1254 and their in vitro-selected imipenem-resistant mutants ISAB ab8(r) and ISAB ab1254(r) were compared, the results showed no change in the bla(OXA-66)/bla(OXA-51-like) gene sequences, in expression of the gene, and in the outer membrane protein profiles. However, there was a four- to eightfold reduction in imipenem resistance upon adding carbonyl cyanide m-chlorophenylhydrazone. Taken together, these results suggest that the OXA-66/OXA-51-like carbapenemase contributes to intrinsic resistance to imipenem; however, drug export by an efflux pump may be more important and/or occur more frequently in imipenem-resistant A. baumannii. Furthermore, this is the first report of a Taiwanese strain of an OXA-66/OXA-51-like carbapenemase that confers imipenem resistance in A. baumannii.

Dataset Information

Structural Basis for Enhancement of Carbapenemase Activity in the OXA-51 Family of Class D ?-Lactamases.

Publications

Structural Basis for Enhancement of Carbapenemase Activity in the OXA-51 Family of Class D β-Lactamases.

Similar Datasets

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