title: A tripeptide deletion in the R2 loop of the class C beta-lactamase enzyme FOX-4 impairs cefoxitin hydrolysis and slightly increases susceptibility to beta-lactamase inhibitors.
abstract: OBJECTIVE
  A natural variant of the AmpC enzyme from Escherichia coli HKY28 with a tripeptide deletion (Gly-286/Ser-287/Asp-288) was recently described. The isolate produced an inhibitor-sensitive AmpC beta-lactamase variant that also conferred higher than usual levels of resistance to ceftazidime in the E. coli host. To demonstrate whether this is true in other class C beta-lactamase enzymes, we deleted the equivalent tripeptide in the FOX-4 plasmid-mediated class C beta-lactamase.
  
  
  METHODS
  By site-directed mutagenesis, we deleted the tripeptide Gly-306/Asn-307/Ser-308 of FOX-4, thus generating FOX-4(DeltaGNS). The enzymes (FOX-4 wild-type and DeltaGNS) were purified and kinetic parameters (kcat, Km, kcat/Km) as well as IC50 values of several beta-lactams were assessed. Modelling studies were also performed.
  
  
  RESULTS
  FOX-4(DeltaGNS) did not increase the catalytic efficiency towards ceftazidime, although it conferred a slight increase in the susceptibility to beta-lactamase inhibitors. There was also a noteworthy decrease in the cefoxitin MIC with the FOX-4(DeltaGNS) mutant (from 512 to 16 mg/L) as well as a 10-fold decrease in kcat/Km towards imipenem, which revealed specific structural features.
  
  
  CONCLUSIONS
  Although deletions in the R2-loop are able to extend the substrate spectrum of class C enzymes, the present results do not confirm this hypothesis in FOX-4. The FOX-4 R2 site would already be wide enough to accommodate antibiotic molecules, and thus any amino acid replacement or deletion at this location would not affect the hydrolytic efficiency towards beta-lactams and would have a less drastic effect on the susceptibility to beta-lactamase inhibitors.
authors.affiliation: Laboratorio de Microbiología-INIBIC, Complejo Hospitalario Universitario La Coruña, Xubias de Arriba s/n, 15006 La Coruña, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Mallo
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Pérez-Llarena
authors.forename: Fréderic
authors.initials: F
authors.lastname: Kerff
authors.forename: Nelson C
authors.initials: NC
authors.lastname: Soares
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
authors.forename: Germán
authors.initials: G
authors.lastname: Bou
mesh_terms: D000595:Amino Acid Sequence; D002440:Cefoxitin / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017384:Sequence Deletion; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D065093:beta-Lactamase Inhibitors; D002440:Cefoxitin; C413413:fox-4 protein, E coli; D001618:beta-Lactamases