|Reference : A Tripeptide Deletion in the Class C beta-Lactamase FOX-4 Enzyme Impairs Cefoxitin Hydro...|
|Scientific journals : Article|
|Life sciences : Biochemistry, biophysics & molecular biology|
|A Tripeptide Deletion in the Class C beta-Lactamase FOX-4 Enzyme Impairs Cefoxitin Hydrolysis and Slightly Increases Susceptibility to beta-Lactamase Inhibitors|
|Mallo, Susana [> >]|
|Pérez-Llarena, Francisco J. [> >]|
|Kerff, Frédéric [Université de Liège - ULg > > Centre d'ingénierie des protéines >]|
|Fernández-Moreira, Esteban [> >]|
|Soares, Nelson C. [> >]|
|Galleni, Moreno [Université de Liège - ULg > Département des sciences de la vie > Macromolécules biologiques >]|
|Bou, German [> >]|
|Journal of Antimicrobial Chemotherapy|
|Oxford University Press|
|Yes (verified by ORBi)|
|[en] 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.
|Centre d'Ingénierie des Protéines - CIP|
|File(s) associated to this reference|
All documents in ORBi are protected by a user license.