Fitness landscape of metallo-b-lactamase-mediated antibiotic resistance

PABLO E. TOMATIS; MARÍA ROCÍO MEINI; 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

Protein evolution is crucial for organismal adaptation and fitness. This complex process relies on a subtle interplay between sequence, structure, functionality and stability. The identification of structural traits acquired through evolution could be valuable for anticipating the molecular features that enhance bacterial resistance to β-lactam antibiotics mediated by the expression of β-lactamases, enzymes whose evolution is continuously challenged by the indiscriminate use of antibiotics. Metallo-β-lactamases (MBLs) are the latest generation among these enzymes, whose structural diversity and broad substrate profile, allows them to inactivate most β-lactam antibiotics. Directed evolution on the MBL BcII from Bacillus cereus yielded an optimized variant, that harbors four mutations remote from the active site, which is able to confer greater antibiotic resistance. Two of these mutations display sign epistasis. One of them improves the hydrolysis rate by stabilizing a catalytic intermediate. The second mutation bestows a more flexible scaffold, resulting in a enzyme with broader substrate spectrum. The fitness landscape of metallo-beta-lactamases is such that the stability threshold has not been exhausted. We have also identified structural determinants of the evolution, therefore this approach can be exploited to design mechanism-based inhibitors with an evolutionary perspective.