title: Mimicking natural evolution in metallo-beta-lactamases through second-shell ligand mutations.
abstract: Metallo-beta-lactamases (MBLs) represent the latest generation of beta-lactamases. The structural diversity and broad substrate profile of MBLs allow them to confer resistance to most beta-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.
authors.affiliation: Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina.
authors.forename: Pablo E
authors.initials: PE
authors.lastname: Tomatis
authors.forename: Rodolfo M
authors.initials: RM
authors.lastname: Rasia
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Segovia
authors.forename: Alejandro J
authors.initials: AJ
authors.lastname: Vila
mesh_terms: D019943:Amino Acid Substitution; D001409:Bacillus cereus / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D003035:Cobalt / Q000737:chemistry; D019020:Directed Molecular Evolution; D019143:Evolution, Molecular; D015723:Gene Library; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics*
chemical_list: D008024:Ligands; D008667:Metalloproteins; D003035:Cobalt; C455979:beta-lactamase BcII; D001618:beta-Lactamases