title: Impact of the E540V amino acid substitution in GyrB of Mycobacterium tuberculosis on quinolone resistance.
abstract: Amino acid substitutions conferring resistance to quinolones in Mycobacterium tuberculosis have generally been found within the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase (GyrA) rather than the B subunit of DNA gyrase (GyrB). To clarify the contribution of an amino acid substitution, E540V, in GyrB to quinolone resistance in M. tuberculosis, we expressed recombinant DNA gyrases in Escherichia coli and characterized them in vitro. Wild-type and GyrB-E540V DNA gyrases were reconstituted in vitro by mixing recombinant GyrA and GyrB. Correlation between the amino acid substitution and quinolone resistance was assessed by the ATP-dependent DNA supercoiling assay, quinolone-inhibited supercoiling assay, and DNA cleavage assay. The 50% inhibitory concentrations of eight quinolones against DNA gyrases bearing the E540V amino acid substitution in GyrB were 2.5- to 36-fold higher than those against the wild-type enzyme. Similarly, the 25% maximum DNA cleavage concentrations were 1.5- to 14-fold higher for the E540V gyrase than for the wild-type enzyme. We further demonstrated that the E540V amino acid substitution influenced the interaction between DNA gyrase and the substituent(s) at R-7, R-8, or both in quinolone structures. This is the first detailed study of the contribution of the E540V amino acid substitution in GyrB to quinolone resistance in M. tuberculosis.
authors.affiliation: Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo 001-0020, Japan.
authors.forename: Hyun
authors.initials: H
authors.lastname: Kim
authors.forename: Chie
authors.initials: C
authors.lastname: Nakajima
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Zeaur
authors.initials: Z
authors.lastname: Rahim
authors.forename: Youn Uck
authors.initials: YU
authors.lastname: Kim
authors.forename: Hiroki
authors.initials: H
authors.lastname: Oguri
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Suzuki
mesh_terms: D019943:Amino Acid Substitution; D000995:Antitubercular Agents / Q000494:pharmacology*; D001483:Base Sequence; D027081:DNA Gyrase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015363:Quinolones / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D015363:Quinolones; D027081:DNA Gyrase