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Crystal structure of carbapenemase OXA-58 from Acinetobacter baumannii.

ABSTRACT: Class D ?-lactamases capable of hydrolyzing last-resort carbapenem antibiotics represent a major challenge for treatment of bacterial infections. Wide dissemination of these enzymes in Acinetobacter baumannii elevated this pathogen to the category of most deadly and difficult to treat. We present here the structure of the OXA-58 ?-lactamase, a major class D carbapenemase of A. baumannii, determined to 1.30-? resolution. Unlike two other Acinetobacter carbapenemases, OXA23 and OXA-24, the OXA-58 enzyme lacks the characteristic hydrophobic bridge over the active site, despite conservation of the residues which participate in its formation. The active-site residues in OXA-58 are spatially conserved in comparison to those in other class D ?-lactamases. Lys86, which activates water molecules during the acylation and deacylation steps, is fully carboxylated in the OXA-58 structure. In the absence of a substrate, a water molecule is observed in the active site of the enzyme and is positioned in the pocket that is usually occupied by the 6?-hydroxyethyl moiety of carbapenems. A water molecule in this location would efficiently deacylate good substrates, such as the penicillins, but in the case of carbapenems, it would be expelled by the 6?-hydroxyethyl moiety of the antibiotics and a water from the surrounding medium would find its way to the vicinity of the carboxylated Lys86 to perform deacylation. Subtle differences in the position of this water in the acyl-enzyme complexes of class D ?-lactamases could ultimately be responsible for differences in the catalytic efficiencies of these enzymes against last-resort carbapenem antibiotics.

SUBMITTER: Smith CA

PROVIDER: S-EPMC4023756 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Crystal structure of carbapenemase OXA-58 from Acinetobacter baumannii.

Smith Clyde A CA Antunes Nuno Tiago NT Toth Marta M Vakulenko Sergei B SB

Antimicrobial agents and chemotherapy 20140127 4

Class D β-lactamases capable of hydrolyzing last-resort carbapenem antibiotics represent a major challenge for treatment of bacterial infections. Wide dissemination of these enzymes in Acinetobacter baumannii elevated this pathogen to the category of most deadly and difficult to treat. We present here the structure of the OXA-58 β-lactamase, a major class D carbapenemase of A. baumannii, determined to 1.30-Å resolution. Unlike two other Acinetobacter carbapenemases, OXA23 and OXA-24, the OXA-58 ...[more]

PMID: 24468777

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Project description:Purpose:A novel variant of OXA-23, named OXA-423, was identified in an Acinetobacter baumannii clinical isolate. The aim of this study was to analyse the resistance phenotype of OXA-423. Methods:The A. baumannii strain WY-0713 was isolated from an intensive care unit patient. PCR was used to detect the bla OXA-23-like genes. Amplifying, cloning and sequencing were performed for the complete bla OXA-23-like. The novel bla OXA-423 and its ancestor bla OXA-23 were cloned into the expression vector pET-28b(+), and transformed into E. coli Rosetta (DE3) for antibiotic susceptibility testing. SDS-PAGE, modified Hodge test and CarbaNP test were used for detecting the expression of OXA-423 and OXA-23. Results:PCR screening of A. baumannii WY-0713 was positive for bla OXA-23-like genes. Sequencing of the PCR product identi?ed a novel bla OXA-23-like, named bla OXA-423 which encoding OXA-423. OXA-423 differed from OXA-23 by a crucial amino acid substitution (Val128Ala). The V128A substitution was located at the conserved active-site motifs SAV of OXA-23. Antibiotic susceptibility testing performed using isogenic E. coli showed that the MICs of E. coli Rosetta (pET-OXA-423) for penicillins and carbapenems were lower (reduced MICs 4-fold to 16-fold) than that of E. coli Rosetta (pET-OXA-23). The MICs of cefotaxime, ceftazidime and aztreonam for both transformants remained the same as the acceptor strain. Moreover, OXA-423 was slightly inhibited by sulbactam, clavulanic acid and tazobactam. SDS-PAGE analysis showed that OXA-423 and OXA-23 were conspicuously expressed. Modified Hodge test and CarbaNP test were positive demonstrated both of them were functional. Conclusion:OXA-423, the first report of an amino acid substitution located at conserved active-site motifs of OXA-23, conferred lower MIC values of penicillins and carbapenems as compared with OXA-23, while without affecting the resistance pro?les of expanded-spectrum cephalosporins and aztreonam.

Dataset Information

Crystal structure of carbapenemase OXA-58 from Acinetobacter baumannii.

Publications

Crystal structure of carbapenemase OXA-58 from Acinetobacter baumannii.

Similar Datasets

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