Evolution and structural plasticity of metallo-beta-lactamases

MARÍA ROCÍO MEINI; PABLO E. TOMATIS; ALEJANDRO J. VILA

San Miguel de Tucumán

Congreso; SAIB 2009 - XLIV Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2009

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Directed Molecular Evolution (DME) strategies are currently exploited to generate stable proteins, improve the catalytic efficiency, shape the substrate specificity of enzymes or design new activities. These studies also provide information on protein evolution. We have shown that DME on the metallo-beta-lactamase BcII from B. cereus gives rise to a variant with enhanced catalytic and resistance traits conferred by four mutations. Two of them, G262S and N70S, located under the active site floor, show a strong sign epistasis, which plays a crucial role in the evolutionary trajectory. The other two, V112A and L250S, are located far away from the active site, and their influence in resistance is not so obvious. Our purpose is to analyze how these mutations are able to enhance fitness and to identify the possible pathways that could lead to the acquisition of larger levels of resistance. We found that one third of the 24 possible evolutionary pathways are accessible. This restriction is mainly due to the fact that mutation G262S acts as a bottleneck in the evolutionary trajectory, providing a more evolvable genetic background than wt BcII. We have also performed DME experiments on inactive mutants of BcII with active side substitutions, which proved reluctant to recover the native activity, suggesting that viable evolutionary pathways proceed by accumulating single base mutations.