β-lactam antibiotics are the most employed chemotherapeutic agents against bacterial infections. The most prevalent resistance mechanism encounter in pathogenic and opportunistic bacteria is the expression of β-lactamases, enzymes able to hydrolyze these antibiotics, rendering them useless. Metallo-β-lactamases (MβLs) are the most recent generation among these proteins. MβLs are broad spectrum enzymes, since they are able to inactivate all β-lactam antibiotics, including carbapenems (the last resource in therapeutic prescription). Genes coding for MβLs are present in mobile genetic elements, to that they have been disseminated world-wide among opportunistic and pathogenic organisms. This picture is aggravated by the structural diversity among MβLs, which makes it difficult to design a pan-MβL inhibitor. In particular, enzyme NDM-1 (http://www.bbc.co.uk/news/health-10925411) has recently been disseminated worldwide, and there are no perspectives of an effective inhibitor, thus representing a threat for global health. Our main goal is to elucidate the structural determinants of function in these enzymes by means of biochemical, structural, mechanistic and evolutionary studies, with the ultimate goal of designing a clinically useful inhibitor. So far, we have been able to: (1) propose a catalytic mechanism for MβLs based on the characterization of a common reaction intermediate, (2) identify the in vivo functional species of MβLs and (3) explore their possible evolutionary pathways. We are now exploiting this knowledge to design new MβL inhibitors, assuming that they share a common catalytic mechanism despite their structural diversity. We currently undertake an interdisciplinary approach, using tools from molecular biology, biochemistry, enzymology and structural biology. Mechanistic studies are performed by using fast-mixing techniques and various spectroscopies to monitor changes in the enzyme active site and in the substrate in a catalytically relevant timescale (miliseconds). We employ protein crystallography and NMR to characterize the binding mode of possible inhibitores. identificar intermediarios de reacción. Utilizamos cristalografía y/o Resonancia Magnética Nuclear (RMN) para caracterizar el modo de unión de posibles inhibidores a estas enzimas. We also use techniques of directed evolution in vitro to anticipate possible evolutionary events of these enzymes, and to understand how different mutations are fixed during evolution.