Dataset Information

Specific Amino Acid Substitutions in OXA-51-Type β-Lactamase Enhance Catalytic Activity to a Level Comparable to Carbapenemase OXA-23 and OXA-24/40.

ABSTRACT: The chromosomal bla_OXA-51-type gene encodes carbapenem-hydrolyzing class D β-lactamases (CHDLs), specific variants shown to mediate carbapenem resistance in the Gram-negative bacterial pathogen Acinetobacter baumannii. This study aims to characterize the effect of key amino acid substitutions in OXA-51 variants of carbapenem-hydrolyzing class D β-lactamases (CHDLs) on substrate catalysis. Mutational and structural analyses indicated that each of the L167V, W222G, or I129L substitutions contributed to an increase in catalytic activity. The I129L mutation exhibited the most substantial effect. The combination of W222G and I129L substitutions exhibited an extremely strong catalytic enhancement effect in OXA-66, resulting in higher activity than OXA-23 and OXA-24/40 against carbapenems. These findings suggested that specific arrangement of residues in these three important positions in the intrinsic OXA-51 type of enzyme can generate variants that are even more active than known CHDLs. Likewise, mutation leading to the W222M change also causes a significant increase in the catalytic activity of OXA-51. bla_OXA-51 gene in A. baumannii may likely continue to evolve, generating mutant genes that encode carbapenemase with extremely strong catalytic activity.

SUBMITTER: Chan KW

PROVIDER: S-EPMC9105447 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Specific Amino Acid Substitutions in OXA-51-Type β-Lactamase Enhance Catalytic Activity to a Level Comparable to Carbapenemase OXA-23 and OXA-24/40.

Chan Kwan-Wai KW Liu Chen-Yu CY Wong Ho-Yin HY Chan Wai-Chi WC Wong Kwok-Yin KY Chen Sheng S

International journal of molecular sciences 20220419 9

The chromosomal blaOXA-51-type gene encodes carbapenem-hydrolyzing class D β-lactamases (CHDLs), specific variants shown to mediate carbapenem resistance in the Gram-negative bacterial pathogen Acinetobacter baumannii. This study aims to characterize the effect of key amino acid substitutions in OXA-51 variants of carbapenem-hydrolyzing class D β-lactamases (CHDLs) on substrate catalysis. Mutational and structural analyses indicated that each of the L167V, W222G, or I129 ...[more]

PMID: 35562886

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Project description:PurposeA 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.MethodsThe 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.ResultsPCR screening of A. baumannii WY-0713 was positive for bla OXA-23-like genes. Sequencing of the PCR product identiﬁed 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.ConclusionOXA-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 proﬁles of expanded-spectrum cephalosporins and aztreonam.

Dataset Information

Specific Amino Acid Substitutions in OXA-51-Type β-Lactamase Enhance Catalytic Activity to a Level Comparable to Carbapenemase OXA-23 and OXA-24/40.

Publications

Specific Amino Acid Substitutions in OXA-51-Type <i>β</i>-Lactamase Enhance Catalytic Activity to a Level Comparable to Carbapenemase OXA-23 and OXA-24/40.

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