title: Role of a mutation at position 167 of CTX-M-19 in ceftazidime hydrolysis.
abstract: CTX-M-19 is a recently identified ceftazidime-hydrolyzing extended-spectrum beta-lactamase, which differs from the majority of CTX-M-type beta-lactamases that preferentially hydrolyze cefotaxime but not ceftazidime. To elucidate the mechanism of ceftazidime hydrolysis by CTX-M-19, the beta-lactam MICs of a CTX-M-19 producer, and the kinetic parameters of the enzyme were confirmed. We reconfirmed here that CTX-M-19 is also stable at a high enzyme concentration in the presence of bovine serum albumin (20 micro g/ml). Under this condition, we obtained more accurate kinetic parameters and determined that cefotaxime (k(cat)/K(m), 1.47 x 10(6) s(-1) M(-1)), cefoxitin (k(cat)/K(m), 62.2 s(-1) M(-1)), and aztreonam (k(cat)/K(m), 1.34 x 10(3) s(-1) M(-1)) are good substrates and that imipenem (k(+2)/K, 1.20 x 10(2) s(-1) M(-1)) is a poor substrate. However, CTX-M-18 and CTX-M-19 exhibited too high a K(m) value (2.7 to 5.6 mM) against ceftazidime to obtain their catalytic activity (k(cat)). Comparison of the MICs with the catalytic efficiency (k(cat)/K(m)) of these enzymes showed that some beta-lactams, including cefotaxime, ceftazidime, and aztreonam showed a similar correlation. Using the previously reported crystal structure of the Toho-1 beta-lactamase, which belongs to the CTX-M-type beta-lactamase group, we have suggested characteristic interactions between the enzymes and the beta-lactams ceftazidime, cefotaxime, and aztreonam by molecular modeling. Aminothiazole-bearing beta-lactams require a displacement of the aminothiazole moiety due to a severe steric interaction with the hydroxyl group of Ser167 in CTX-M-19, and the displacement affects the interaction between Ser130 and the acidic group such as carboxylate and sulfonate of beta-lactams.
authors.affiliation: Department of Microbiology, 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: Masaji
authors.initials: M
authors.lastname: Ishiguro
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Ishii
authors.forename: Jimena
authors.initials: J
authors.lastname: Alba
authors.forename: Keizo
authors.initials: K
authors.lastname: Yamaguchi
mesh_terms: D001398:Aztreonam / Q000494:pharmacology; D002442:Ceftazidime / Q000378:metabolism*; D002511:Cephalosporins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008997:Monobactams / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology*; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002511:Cephalosporins; D008997:Monobactams; D002442:Ceftazidime; D001618:beta-Lactamases; C486443:beta-lactamase CTX-M-19; D001398:Aztreonam