Dataset Information

Expression, crystallization and preliminary X-ray crystallographic analysis of cystathionine ?-lyase from Acinetobacter baumannii OXA-23.

ABSTRACT: Multidrug-resistant Acinetobacter baumannii (Ab) has emerged as a leading nosocomial pathogen because of its resistance to most currently available antibiotics. Cystathionine ?-lyase (CBL), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the second step in the transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids homocysteine and methionine. The enzymes of the transsulfuration pathway are considered to be attractive drug targets owing to their specificity to microbes and plants. As a potential target for the development of novel antibacterial drugs, the AbCBL protein was expressed, purified and crystallized. An AbCBL crystal diffracted to 1.57?Å resolution and belonged to the trigonal space group P3112, with unit-cell parameters a = b = 102.9, c = 136.5?Å. The asymmetric unit contained two monomers, with a corresponding VM of 2.3?Å(3)?Da(-1) and a solvent content of 46.9%.

SUBMITTER: Nguyen DQ

PROVIDER: S-EPMC4188081 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Expression, crystallization and preliminary X-ray crystallographic analysis of cystathionine β-lyase from Acinetobacter baumannii OXA-23.

Nguyen Diem Quynh DQ Ngo Ho Phuong Thuy HP Ahn Yeh Jin YJ Lee Sang Hee SH Kang Lin Woo LW

Acta crystallographica. Section F, Structural biology communications 20140925 Pt 10

Multidrug-resistant Acinetobacter baumannii (Ab) has emerged as a leading nosocomial pathogen because of its resistance to most currently available antibiotics. Cystathionine β-lyase (CBL), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the second step in the transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids homocysteine and methionine. The enzymes of the transsulfuration pathway are considered to be attractive drug ta ...[more]

PMID: 25286941

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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

Expression, crystallization and preliminary X-ray crystallographic analysis of cystathionine ?-lyase from Acinetobacter baumannii OXA-23.

Publications

Expression, crystallization and preliminary X-ray crystallographic analysis of cystathionine β-lyase from Acinetobacter baumannii OXA-23.

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