Unveiling fitness evolution mechanisms for metallo-beta-lactamases

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

Salta

Congreso; LAPSM 2010- 3rd Latin American Protein Society Meeting; 2010

Latin American Protein Society

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. 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 for fitness. G262S increases the enzyme catalytic efficiency and N70S, the flexibility of the active site. The other two, V112A and L250S, are located far away from the active site, and their influence on the enzyme function is not so obvious. We analyzed how these mutations are able to enhance fitness and we identified the possible pathways that could lead to the acquisition of larger levels of resistance. Interestingly, only 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, providing a more evolvable genetic background than wt BcII. However, the increase in the enzyme activity was not enough to explain its enhanced capacity to confer resistance. We have found that V112A acts as a stabilizing mutation; therefore, we can conclude that stability is another contributing factor to the enzyme fitness.