Dissecting evolutionary traits in Zinc beta-lactamases

ALEJANDRO JOSE VILA

Rio de Janeiro

Congreso; LAPS 2d Latin American Protein Society Acapulco; 2007

Metallo-beta-lactamases (MBLs) represent the latest generation of beta-lactamases. Their structural diversity and broad substrate profile allows them to inactivate most beta-lactam antibiotics. Directed evolution on the Bacillus cereus MBL, BcII, yielded an optimized variant (M5) able to confer more resistance than the native enzyme. This variant harbors four mutations remote from the active site that contribute to broaden its substrate repertoire. Two of these mutations display an epistatic effect: whereas one them accelerates the rate-limiting step of the mechanism by stabilizing a catalytic intermediate, the second one bestows a more dynamic scaffold to counterbalance the effect of the previous mutation, resulting in a broader substrate spectrum. These optimizations take place at the expense of the protein stability. This reveals that MBLs are still incipient enzymes within the bacterial resistance machinery, since they are still able to evolve by optimizing substrate recognition and transition state stabilization, and that this effect can be achieved by a network of coupled motions.