Catalytic mechanism and evolutionary traits of Zn-dependent beta-lactamases

MARÍA ROCÍO MEINI; MARIANO M. GONZÁLEZ; LISANDRO J. GONZÁLEZ; GUILLERMO BAHR; ANTONELA PALACIOS; LETICIA I. LLARRULL; ALEJANDRO J. VILA

Punta del Este

Simposio; XII Symposium on Metal Ions in Biology and Medicine; 2013

Química Inorgánica, DEC Facultad de Química, Universidad de la República Uruguay

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 Zn(II)-dependent enzymes. The exponential growth of MBL sequences being characterized has revealed an initially unforeseen structural diversity, that gives rise to the presence of mono- and dinuclear metal sites. MBL´s have been recently 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 means of 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 b-lactam hydrolysis, and to assess the role of each metal binding site in the mechanism and stabilization of this intermediate. 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. These results suggest that evolution of the chemical landscape can be predicted by means of this approach. We have also observed that evolution acts by enhancing the Zn(II) binding capability upon under metal-defficient conditions.