Structure, Catalysis and Evolution of zinc beta-lactamases

VILA, A J

Buzios

Congreso; VII Iberoamerican Congress of Biophysics; 2009

Sociedad de Biofisica latinoamericana

b-lactamases represent the prevalent resistance mechanism to b-lactam antibiotics. In the last decade, the dissemination of genes coding for metallo-b-lactamases (MBL´s) has become an emergent clinical problem. MBL´s are zinc-dependent enzymes. They display an initially unforeseen structural diversity, that gives rise to the presence of mono- and dinuclear metal sites. MBL´s have been subdivided into classes B1, B2 and B3, each of them displaying different zinc ligands and coordination geometries. We have studied the structural features of MBL´s from different subclasses with the aim of finding common structural and catalytic features. By mutagenesis, functional and structural studies, we conclude that a Zn site, previously regarded as non essential for catalysis, plays a major role in substrate binding and catalysis. Non-steady state kinetic studies, aided by time-resolved electronic, EPR and Resonance Raman spectroscopy have allowed us to trap a key intermediate in β-lactam hydrolysis, and to assess the role of each metal binding site in the mechanism and stabilization of this intermediate. Finally, directed evolution was used as an evolutionary engineering tool to explore the effect of challenging MBLs towards different antibiotics. BcII (the MBL from B.cereus) has been considered as a precursor of more efficient MBL’s present in pathogenic bacteria. In vitro evolution experiments on BcII by DNA shuffling with a cephalosporin substrate resulted in a expanded substrate spectrum of this enzyme, without sacrificing its stability nor the hydrolytic efficiency towards classical substrates of BcII. The mutations that give rise to these effects parallel others naturally found in MBL´s from pathogenic bacteria, and are related to the second-shell ligands of the zinc ions, expected to play a supramolecular control of reactivity. These results suggest that evolution of the chemical landscape can be predicted by means of this approach.