Dramatic broadening of the substrate profile of the Aeromonas hydrophila CphA metallo-beta-lactamase by site-directed mutagenesis

Among class B beta-lactamases, the subclass B2 CphA enzyme is characterized by a unique specificity profile. CphA efficiently hydrolyzes only carbapenems. In this work, we generated site-directed mutants that possess a strongly broadened activity spectrum when compared with the WT CphA. Strikingly, the N116H/N220G double mutant exhibits a substrate profile close to that observed for the broad spectrum subclass B1 enzymes. The double mutant is significantly activated by the binding of a second zinc ion under conditions where the WT enzyme is non-competitively inhibited by the same ion.