Metallo-beta-lactamases: antibiotic resistance and the role of periplasmic Zn(II)

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

Lanzarote

Conferencia; Zing Conference on Bioinorganic Chemistry; 2013

Zing Confereces

Beta-lactamases represent the prevalent resistance mechanism to beta-lactam antibiotics. In the 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. 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 (1). 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 (2-5, 10, 11). Non-steady state kinetic studies, aided by time-resolved electronic, EPR and Resonance 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 mechanism and stabilization of this intermediate (5,6). Finally, directed evolution was used as an evolutionary engineering tool to explore the effect of challenging MBLs towards different antibiotics. 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 (7,8). 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. Moreover, we found that zinc binding is limiting within the bacterial periplasm to elicit resistance and can be tuned during evolution. References 1. M. W. Crowder, J. Spencer and A.J. Vila. Acc. Chem.Res, 2006,39,721. 2. Llarrull et al. J. Biol.Chem., 2007, 282, 18276. 3. Morán-Barrio et al. J.Biol.Chem., 2007, 282, 18286. 4. Llarrull et al. J.Biol.Chem., 2007, 282, 30586. 5. Tioni et al. J.Am.Chem.Soc., 2008, 130, 15852. 6. Llarrull et al. J.Am.Chem.Soc., 2008, 130, 15842. 7. Tomatis et al. Proc.Natl.Acad.Sci.USA, 2005, 102, 13761. 8. Tomatis et al. Proc.Natl.Acad.Sci.USA, 2008, 105, 20605. 9. Gonzalez et al. Nature Chem. Biol., 2012, 8, 698. 10. Morán-Barrio et al. Antimicrobial Agents and Chem., 2012, 56, 1769.