Abstract
Metallo-beta-lactamases are zinc enzymes able to hydrolyze the four-membered ring of beta-lactam antibiotics, representing one of the latest generations of beta-lactamases. These enzymes belong to the zinc metallo-hydrolase family of the beta-lactamase fold. Enzymes belonging to this family have a bimetallic active site whose structure varies among different members by point substitutions of the metal ligands. In this work, we have grafted new metal ligands into the metal binding site of BcII from Bacillus cereus that mimic the ligands present in other members of this superfamily. We have characterized spectroscopically and modeled the structure of the redesigned sites, which differ substantially from the wild-type enzyme. Despite the changes introduced in the active site, the mutant enzymes retain almost full activity. These results shed some light on the possible evolutionary origin of these metalloenzymes.