Catalytic Mechanism and Evolutionary traits of zinc beta-lactamases: Does it take two to tango?

A. J. VILA

Simposio; XI International Symposium on Inorganic Biochemistry; 2010

Beta-lactamases represent the prevalent resistance mechanism to beta-lactam antibiotics. Inthe last decade, the dissemination of genes coding for metallo-beta-lactamases (MBL´s)has become an emergent clinical problem. MBL´s are zinc-dependent enzymes. Theexponential growth of MBL sequences being characterized has revealed an initiallyunforeseen structural diversity, that gives rise to the presence of mono- and dinuclearmetal sites. MBL´s have been recently subdivided into classes B1, B2 and B3, each ofthem displaying different zinc ligands and coordination geometries (1).We have studied the structural features of MBL´s from different subclasses with the aimof finding common structural and catalytic features. By means of mutagenesis,functional and structural studies, we conclude that a Zn site, previously regarded asnon essential for catalysis, plays a major role in substrate binding and catalysis (2-5).Non-steady state kinetic studies, aided by time-resolved electronic, EPR andResonance Raman spectroscopy have allowed us to trap a key intermediate in beta-lactam hydrolysis, and to assess the role of each metal binding site in the mechanismand stabilization of this intermediate (6-8).Finally, directed evolution was used as an evolutionary engineering tool to explore theeffect 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 pathogenicbacteria. In vitro evolution experiments on BcII by DNA shuffling with a cephalosporinsubstrate resulted in a expanded substrate spectrum of this enzyme, without sacrificingits stability nor the hydrolytic efficiency towards classical substrates of BcII (9,10). Themutations that give rise to these effects parallel others naturally found in MBL´s frompathogenic bacteria, and are related to the second-shell ligands of the zinc ions,expected to play a supramolecular control of reactivity. These results suggest thatevolution of the chemical landscape can be predicted by means of this approach.