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Achieving Regio- and Enantioselectivity of P450-Catalyzed Oxidative CH Activation of Small Functionalized Molecules by Structure-Guided Directed Evolution

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Agudo Torres,  Rubén
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Philipps-Universität Marburg, Fachbereich Chemie;

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Roiban,  Georghe-Doru
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Philipps-Universität Marburg, Fachbereich Chemie;

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Reetz,  Manfred T.
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Philipps-Universität Marburg, Fachbereich Chemie;

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Citation

Agudo Torres, R., Roiban, G.-D., & Reetz, M. T. (2012). Achieving Regio- and Enantioselectivity of P450-Catalyzed Oxidative CH Activation of Small Functionalized Molecules by Structure-Guided Directed Evolution. ChemBioChem: A European Journal of Chemical Biology, 13(10), 1465-1473. doi:10.1002/cbic.201200244.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0010-510A-6
Abstract
Directed evolution of the monooxygenase P450-BM3 utilizing iterative saturation mutagenesis at and near the binding site enables a high degree of both regio- and enantioselectivity in the oxidative hydroxylation of cyclohexene-1-carboxylic acid methyl ester. Wild-type P450-BM3 is 84% regioselective for the allylic 3-position with 34% enantioselectivity in favor of the R alcohol. Mutants enabling R selectivity (>95% ee) or S selectivity (>95% ee) were evolved, while reducing other oxidation products and thus maximizing regioselectivity to >93%. Control of the substrate-to-enzyme ratio is necessary for obtaining optimal and reproducible enantioselectivities, an observation which is important in future protein engineering of these mono-oxygenases. An E. coli strain capable of NADPH regeneration was also engineered, simplifying directed evolution of P450 enzymes in general. These synthetic results set the stage for subsequent stereoselective and stereospecific chemical transformations to form more complex compounds, thereby illustrating the viability of combining genetically altered enzymes as catalysts in organic chemistry with traditional chemical methods.