title: Predictive analysis of ceftazidime hydrolysis in CTX-M-type beta-lactamase family members with a mutational substitution at position 167.
abstract: The CTX-M family of extended-spectrum beta-lactamases has been increasing in number over recent years. Its members preferentially hydrolyse cefotaxime over ceftazidime. Recently, ceftazidime-hydrolysing CTX-M beta-lactamase producers with a mutation at Pro167Ser have been found. The aim of this study was to determine whether members of the CTX-M-type beta-lactamase family are capable of ceftazidime hydrolysis after introduction of the Pro167Ser point mutation. MICs of wild-type enzyme producers for cefotaxime were 2-4 times higher than those of their respective Pro167Ser mutants, whereas MICs of wild-type enzyme producers for ceftazidime were 4-32 times lower than those of their respective Pro167Ser mutants. The k(cat)/K(m) values for Pro167Ser mutants and their respective wild-type enzymes were identical for cefalothin, penicillin and nitrocefin. For cefotaxime, catalytic efficiency (k(cat)/K(m)) for wild-type enzymes was 3.13-7.12 times higher than that of their respective Pro167Ser mutants. As these enzymes exhibit a very high K(m) value (>680 mM) for ceftazidime, we measured initial hydrolysis rates for each enzyme at a low substrate concentration (10 microM) to obtain their k(cat) and k(cat)/K(m) values. Under these conditions, Pro167Ser mutants had k(cat)/K(m) values 1.73-2.21 times higher than those of their respective wild-type enzymes. These results indicate that the CTX-M-type beta-lactamase family can hydrolyse ceftazidime more efficiently because of the point mutation at position 167.
authors.affiliation: Department of Microbiology and Infectious Diseases, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 1438540, Japan.
authors.forename: Soichiro
authors.initials: S
authors.lastname: Kimura
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Ishii
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Tateda
authors.forename: Keizo
authors.initials: K
authors.lastname: Yamaguchi
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002442:Ceftazidime / Q000378:metabolism* / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D001618:beta-Lactamases / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D002442:Ceftazidime; D001618:beta-Lactamases