Evolution, diversity, and catalyitc mechanism of metallo-beta-lactamases

TOMATIS, P.E.; LLARRULL, L.I.; TIONI, M.F.; GONZÁLEZ, J. M.; CAMPOS BERMÚDEZ, V.A.; GONZÁLEZ, L.; ABRIATA, L.A.; VILA, A.J.

Mérida

Congreso; 2005 Meeting of International Research Scholars, Howard Hughes Medical Institute; 2005

Howard Hughes Medical Institute

Beta-lactam antibiotics are one of the cornerstones of antibacterial chemiotherapy. The increasing use of these drugs in clinical settings places a high selective pressure that favors selection of pathogenic microorganisms through the development of different resistance mechanisms, one of which is the expression of beta-lactamases that inactivate beta-lactam antibiotics. Directed evolution has been put forward as a powerful tool to predict antibiotic resistance. We have used this strategy as a tool of evolutionary engineering to explore the effect of challenging the metallo-beta-lactamase (MBL) BcII from Bacillus cereus towards different antibiotics. This enzyme has been considered as precursor of more efficient MBLs present in pathogenic bacteria. We have performed in vitro evolution experiments of BcII by DNA shuffling with a cephalosporin substrate, which resulted in an expanded substrate spectrum of this enzyme without sacrificing its stability or the hydrolytic efficiency towards classical substrates of BcII. Some of these mutations, in second-shell ligands of the metal ions, have been found in related enzymes from pathogens, revealing that we have been able to mimic natural evolution. We intend to explore this information to predict future mutations able to confer resistance.