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Learning from Directed Evolution: Further Lessons from Theoretical Investigations into Cooperative Mutations in Lipase Enantioselectivity
Author(s) -
Reetz Manfred T.,
Puls Michael,
Carballeira José Daniel,
Vogel Andreas,
Jaeger KarlErich,
Eggert Thorsten,
Thiel Walter,
Bocola Marco,
Otte Nikolaj
Publication year - 2007
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200600359
Subject(s) - mutant , enantioselective synthesis , cooperativity , directed evolution , kinetic resolution , chemistry , lipase , mutation , decanoic acid , stereochemistry , biochemistry , genetics , catalysis , biology , enzyme , gene
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).