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Objectives: 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([DELTA]GNS). The enzymes (FOX-4 wild-type and [DELTA]GNS) 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([DELTA]GNS) 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([DELTA]GNS) 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.

(C) British Society for Antimicrobial Chemotherapy 2010. Published by Oxford University Press. All rights reserved.