title: Structure-function studies of arginine at position 276 in CTX-M beta-lactamases.
abstract: OBJECTIVE
  In order to assess whether or not the Arg-276 of CTX-M-type enzymes is equivalent to the Arg-244 of IRT-TEM-derivative enzymes, we replaced the former with six different amino acids, some of them previously described as involved in resistance to beta-lactamase inhibitors in TEM-IRT derivatives. We also investigated the role of Arg276 in cefotaxime hydrolysis.
  
  
  METHODS
  By site-directed mutagenesis and by use of the bla(CTX-M-1) gene as template, Arg-276 was replaced with six different amino acids (Trp, His, Cys, Asn, Gly and Ser). MICs of beta-lactams alone and in combination with beta-lactamase inhibitors were established. The seven enzymes (CTX-M-1 wild-type and six derived mutants) were purified by affinity chromatography, and kinetic parameters (k(cat), K(m), k(cat)/K(m)) towards cefalotin and cefotaxime were determined. Clavulanic acid IC(50) values were also assessed with all enzymes.
  
  
  RESULTS
  No increase in MICs of beta-lactam/beta-lactamase inhibitor combination was detected with any of the six CTX-M-1-derived mutants, in agreement with the clavulanic acid IC(50) values. The MICs of cefotaxime were clearly lower for the Escherichia coli harbouring the Trp, Cys, Ser and Gly CTX-M-1 mutant enzymes than for CTX-M-1, and these enzymes showed a clearly reduced catalytic efficiency towards cefotaxime. As regards cefalotin, there was a moderate reduction in catalytic efficiency for Cys and His.
  
  
  CONCLUSIONS
  Arg-276 in CTX-M-type beta-lactamases is not equivalent to Arg-244 in IRT-type enzymes. Position Arg-276 appears to be important for cefotaxime hydrolysis in CTX-M-type enzymes, although different effects were obtained regarding the replaced amino acid.
authors.affiliation: Servicio de Microbiología, Unidad de Investigación, Complejo Hospitalario Universitario Juan Canalejo, La Coruña, Spain.
authors.forename: Francisco José
authors.initials: FJ
authors.lastname: Pérez-Llarena
authors.forename: Mónica
authors.initials: M
authors.lastname: Cartelle
authors.forename: Susana
authors.initials: S
authors.lastname: Mallo
authors.forename: Alejandro
authors.initials: A
authors.lastname: Beceiro
authors.forename: Astrid
authors.initials: A
authors.lastname: Pérez
authors.forename: Rosa
authors.initials: R
authors.lastname: Villanueva
authors.forename: Antonio
authors.initials: A
authors.lastname: Romero
authors.forename: Richard
authors.initials: R
authors.lastname: Bonnet
authors.forename: Germán
authors.initials: G
authors.lastname: Bou
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D002439:Cefotaxime / Q000378:metabolism / Q000494:pharmacology; D002512:Cephalothin / Q000378:metabolism / Q000494:pharmacology; D002846:Chromatography, Affinity; D019818:Clavulanic Acid / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D019818:Clavulanic Acid; C055184:beta-lactamase TEM-3; D001618:beta-Lactamases; D002439:Cefotaxime; D002512:Cephalothin