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Directed Evolution of an Enantioselective Enoate‐Reductase: Testing the Utility of Iterative Saturation Mutagenesis
Author(s) -
Bougioukou Despina J.,
Kille Sabrina,
Taglieber Andreas,
Reetz Manfred T.
Publication year - 2009
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.200900644
Subject(s) - saturated mutagenesis , chemistry , directed evolution , enantioselective synthesis , mutagenesis , biocatalysis , stereochemistry , protein engineering , substrate (aquarium) , oxidoreductase , combinatorial chemistry , mutant , catalysis , enzyme , biochemistry , gene , reaction mechanism , oceanography , geology
Abstract Directed evolution utilizing iterative saturation mutagenesis (ISM) has been applied to the old yellow enzyme homologue YqjM in the quest to broaden its substrate scope, while controlling the enantioselectivity in the bioreduction of a set of substituted cyclopentenone and cyclohexenone derivatives. Guided by the known crystal structure of YqjM, 20 residues were selected as sites for saturation mutagenesis, a pooling strategy based on the method of Phizicky [M. R. Martzen, S. M. McCraith, S. L. Spinelli, F. M. Torres, S. Fields, E. J. Grayhack, E. M. Phizicky, Science 1999 , 286 , 1153–1155] being used in the GC screening process. The genes of some of the hits were subsequently employed as templates for randomization experiments at the other putative hot spots. Both ( R )‐ and ( S )‐selective variants were evolved using 3‐methylcyclohexenone as the model substrate in the asymmetric bioreduction of the olefinic functionality, only small mutant libraries and thus minimal screening effort being necessary. Some of the best mutants also proved to be excellent catalysts when testing other prochiral substrates without resorting to additional mutagenesis/screening experiments. Thus, the results constitute an important step forward in generalizing the utility of ISM as an efficient method in laboratory evolution of enzymes as catalysts in organic chemistry.