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

作者：Mallo Susana; Perez Llarena Francisco J; Kerff Frederic; Soares Nelson C; Galleni Moreno; Bou German^*

来源：Journal of Antimicrobial Chemotherapy, 2010, 65(6): 1187-1194.

DOI：10.1093/jac/dkq115

• plasmid-mediated AmpC
• cephamycinase
• R2-loop
• structure-function

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.
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 (k(cat), K-m, k(cat)/K-m) as well as IC50 values of several beta-lactams were assessed. Modelling studies were also performed.
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 k(cat)/K-m towards imipenem, which revealed specific structural features.
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.

• 出版日期2010-6