Effect of remote and second shell mutations in enzymes from the metallo-beta-lactamase superfamily

ALEJANDRO JOSE VILA

Montevideo

Congreso; 6th International Conference of Biological Physics; 2007

ICBP

Metallo-beta-lactamases (MBLs) are zinc-dependent enzymes able to hydrolyze all beta-lactam based drugs. They belong to a large superfamily of metalloenzymes, mostly hydrolases, that employ different metal ions for their biological roles. Sequence analysis of MBLs reveals a large structural diversity, that gives rise to the presence of active sites with one and two metal ions. MBLs also differ from the other members of the large superfamily by the metal ligands.        Engineering the ligand set of other members of the superfamily into the MBL from B.cereus (BcII) results in an enzyme with sensibly impaired catalytic features, that is due to a subtle shift in the position of one metal ion in the catalytic center.        Directed evolution on BcII by DNA shuffling resulted in a expanded substrate spectrum of this enzyme, without sacrificing its stability nor the hydrolytic efficiency towards classical substrates of BcII. The mutations that give rise to these effects parallel others naturally found in MBL´s from pathogenic bacteria, and are related to the second-shell ligands of the second zinc ion, expected to play a supramolecular control of reactivity. We have also studied glyoxalase II from S. typhimurium, a Fe-dependent hydrolase that belongs to the MBL superfamily. Based on the crystal structure of this enzyme, we propose that the metal ion selectivity is controled by a second-shell ligand.