title: Roles of residues Cys69, Asn104, Phe160, Gly232, Ser237, and Asp240 in extended-spectrum beta-lactamase Toho-1.
abstract: Toho-1, which is also designated CTX-M-44, is an extended-spectrum class A β-lactamase that has high activity toward cefotaxime. In this study, we investigated the roles of residues suggested to be critical for the substrate specificity expansion of Toho-1 in previous structural analyses. Six amino acid residues were replaced one by one with amino acids that are often observed in the corresponding position of non-extended-spectrum β-lactamases. The mutants produced in Escherichia coli strains were analyzed both for their kinetic properties and their effect on drug susceptibilities. The results indicate that the substitutions of Asn104 and Ser237 have certain effects on expansion of substrate specificity, while those of Cys69 and Phe160 have less effect, and that of Asp240 has no effect on the hydrolysis of any substrates tested. Gly232, which had been assumed to increase the flexibility of the substrate binding site, was revealed not to be critical for the expansion of substrate specificity of this enzyme, although this substitution resulted in deleterious effects on expression and stability of the enzyme.
authors.affiliation: Department of Nutritional Sciences, Faculty of Applied Bio-Science, Tokyo University of Agriculture, Tokyo, Japan. aibuka@mail.ecc.u-tokyo.ac.jp
authors.forename: Akiko
authors.initials: A
authors.lastname: Shimizu-Ibuka
authors.forename: Mika
authors.initials: M
authors.lastname: Oishi
authors.forename: Shoko
authors.initials: S
authors.lastname: Yamada
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Ishii
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Mura
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakai
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuzawa
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001665:Binding Sites; D002439:Cefotaxime / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001618:beta-Lactamases; D002439:Cefotaxime