title: Learning from directed evolution: Further lessons from theoretical investigations into cooperative mutations in lipase enantioselectivity.
abstract: An earlier experimental study, which involved the directed evolution of enantioselective lipase variants from Pseudomonas aeruginosa as catalysts in the hydrolytic kinetic resolution of 2-methyl-decanoic acid p-nitrophenyl ester, provided a mutant with six mutations. Consequently, the selectivity factor was found to increase from E = 1.1 for the wild-type to E = 51 for the best mutant. Only one of the amino acid exchanges in this mutant was found to occur next to the binding pocket, the other mutations being remote. Our previous theoretical analysis with molecular-dynamics simulations helped to unveil the source of enhanced enantioselectivity: a relay mechanism that involves two of the six mutations was shown to induce strong cooperativity. In this investigation, single, double, and triple mutants were constructed and tested as enantioselective catalysts. This study supports our original postulate regarding the relay mechanism, offers further mechanistic insight into the role of individual mutations, and provides mutants that display even higher enantioselectivity (E of up to 64).
authors.affiliation: Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany. reetz@mpi-muelheim.mpg.de
authors.forename: Manfred T
authors.initials: MT
authors.lastname: Reetz
authors.forename: Michael
authors.initials: M
authors.lastname: Puls
authors.forename: José Daniel
authors.initials: JD
authors.lastname: Carballeira
authors.forename: Andreas
authors.initials: A
authors.lastname: Vogel
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
authors.forename: Thorsten
authors.initials: T
authors.lastname: Eggert
authors.forename: Walter
authors.initials: W
authors.lastname: Thiel
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.forename: Nikolaj
authors.initials: N
authors.lastname: Otte
mesh_terms: D002384:Catalysis; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000737:chemistry; D019020:Directed Molecular Evolution; D004952:Esters; D005821:Genetic Techniques; D007700:Kinetics; D008049:Lipase / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D012984:Software; D013237:Stereoisomerism
chemical_list: D017931:DNA Primers; D004952:Esters; D008049:Lipase