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(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.