Protein evolution is crucial for organismal adaptation and fitness. This process takes place by shaping a given 3-dimensional fold for its particular biochemical function within the metabolic requirements and constraints of the environment. The complex interplay between sequence, structure, functionality, and stability that gives rise to a particular phenotype has limited the identification of traits acquired through evolution. This is further complicated by the fact that mutations are pleiotropic, and interactions between mutations are not always understood. Antibiotic resistance mediated by ␤-lactamases represents an evolutionary paradigm in which organismal fitness depends on the catalytic efficiency of a single enzyme. Based on this, we have dissected the structural and mechanistic features acquired by an optimized metallo-␤-lactamase (M␤L) obtained by directed evolution. We show that antibiotic resistance mediated by this enzyme is driven by 2 mutations with sign epistasis. One mutation stabilizes a catalytically relevant intermediate by fine tuning the position of 1 metal ion; whereas the other acts by augmenting the protein flexibility. We found that enzyme evolution (and the associated antibiotic resistance) occurred at the expense of the protein stability, revealing that M␤Ls have not exhausted their stability threshold. Our results demonstrate that flexibility is an essential trait that can be acquired during evolution on stable protein scaffolds. Directed evolution aided by a thorough characterization of the selected proteins can be successfully used to predict future evolutionary events and design inhibitors with an evolutionary perspective.antibiotic resistance ͉ enzyme ͉ fitness landscape ͉ metalloproteins ͉ epistasis P rotein evolution is crucial for organismal adaptation and fitness (1-8). This process takes place by shaping a given 3-dimensional fold for its particular biochemical function within the metabolic requirements and constraints of the environment.

Metallo-␤-lactamases (MBLs) represent the latest generation of ␤-lactamases. The structural diversity and broad substrate profile of MBLs allow them to confer resistance to most ␤-lactam antibiotics. To explore the evolutionary potential of these enzymes, we have subjected the Bacillus cereus MBL (BcII) to a directed evolution scheme, which resulted in an increased hydrolytic efficiency toward cephalexin. A systematic study of the hydrolytic profile, substrate binding, and active-site features of the evolved lactamase reveal that directed evolution has shaped the active site by means of remote mutations to better hydrolyze cephalosporins with small, uncharged C-3 substituents. One of these mutations is found in related enzymes from pathogenic bacteria and is responsible for the increase in that enzyme's hydrolytic profile. The mutations lowered the activation energy of the rate-limiting step rather than improved the affinity of the enzyme toward these substrates. The following conclusions can be made: (i) MBLs are able to expand their substrate spectrum without sacrificing their inherent hydrolytic capabilities; (ii) directed evolution is able to mimic mutations that occur in nature; (iii) the metal-ligand strength is tuned by second-shell mutations, thereby influencing the catalytic efficiency; and (iv) changes in the position of the second Zn(II) ion in MBLs affect the substrate positioning in the active site. Overall, these results show that the evolution of enzymatic catalysis can take place by remote mutations controlling reactivity.Co(II)-substitution ͉ directed evolution ͉ zinc enzymes ͉ antibiotic resistance T he ␤-lactam antibiotics account for more than half of the world's antibiotic market and are one of the cornerstones of antibacterial chemotherapy (1). The increasing use of these drugs, both in the clinical setting and in animal feed, induces the development of different resistance mechanisms in pathogenic and opportunistic microorganisms (2, 3). Among them, the expression of ␤-lactamases is predominant. In the last decade, there has been growing concern about the dissemination of genes coding for metallo-␤-lactamases (MBLs) among infectious microorganisms (4-7). MBLs are zinc-dependent enzymes that hydrolyze ␤-lactams by favoring the deprotonation of a metalbound water molecule, which (as a hydroxide) is a powerful nucleophile (8-11). Much recent literature has been devoted to the elucidation of the structure and mechanism of action of . Parallel to these efforts, the significant growth of new MBL sequences has revealed an initially unforeseen molecular and structural diversity in this family of enzymes (6). This diversity encompasses changes in the coordination features of the Zn(II) ions as well as in the topology of the active site. This fact has hitherto thwarted the successful development of clinically useful inhibitors. The potential danger of MBLs is further enhanced by the broad substrate spectrum displayed by these enzymes, as compared with the better-characterized Serdependent ␤-lactamas...