The evolution pathway of beta-lactamases

ALEJANDRO J, VILA

San Diego

Congreso; ICAAC; 2015

ICAAC

b-lactamases represent the prevalent resistance mechanism to b-lactamantibiotics. 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-dependentenzymes. The exponential growth of MBL sequences being characterized hasrevealed an initially unforeseen structural diversity, that gives rise to thepresence of mono- and dinuclear metal sites. MBL´s have been recentlysubdivided into classes B1, B2 and B3, each of them displaying different zincligands and coordination geometries.1We have studied the structuralfeatures of MBL´s from different subclasses with the aim of finding commonstructural and catalytic features. By means of mutagenesis, functional andstructural studies, we conclude that a Zn site, previously regarded as nonessential for catalysis, plays a major role in substrate binding and catalysis.2-5,10,11Non-steady state kinetic studies,aided by time-resolved electronic, EPR and Resonance Raman spectroscopy haveallowed us to trap a key intermediate in β-lactam hydrolysis, and to assess therole of each metal binding site in the mechanism and stabilization of thisintermediate.5,6Finally, directed evolution was usedas an evolutionary engineering tool to explore the effect of challenging MBLstowards different antibiotics. In vitroevolution experiments on BcII by DNA shuffling with a cephalosporin substrateresulted in a expanded substrate spectrum of this enzyme, without sacrificingits stability nor the hydrolytic efficiency towards classical substrates ofBcII.7,8 The mutations that give rise to these effects parallelothers naturally found in MBL´s from pathogenic bacteria, and are related tothe second-shell ligands of the zinc ions, expected to play a supramolecularcontrol of reactivity. Moreover, we found that zinc binding is limiting withinthe bacterial periplasm to elicit resistance and can be tuned during evolution.9