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Journal Article

P450-Catalyzed Regio- and Diastereoselective Steroid Hydroxylation: Efficient Directed Evolution Enabled by Mutability Landscaping

MPS-Authors
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Acevedo-Rocha,  Carlos G.
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Department of Chemistry, Philipps-University;

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Lonsdale,  Richard
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Department of Chemistry, Philipps-University;
Centre for Computational Chemistry, School of Chemistry, University of Bristol;

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Li,  Aitao
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Department of Chemistry, Philipps-University;
Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University;

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Lingnau,  Julia B.
Service Department Farès (NMR), Max-Planck-Institut für Kohlenforschung, Max Planck Society;

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Wirtz,  Cornelia
Service Department Farès (NMR), Max-Planck-Institut für Kohlenforschung, Max Planck Society;

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Fares,  Christophe
Service Department Farès (NMR), Max-Planck-Institut für Kohlenforschung, Max Planck Society;

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Hinrichs,  Heike
Service Department Schulze (GC, HPLC), Max-Planck-Institut für Kohlenforschung, Max Planck Society;

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Deege,  Alfred
Service Department Schulze (GC, HPLC), Max-Planck-Institut für Kohlenforschung, Max Planck Society;

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Reetz,  Manfred T.
Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society;
Department of Chemistry, Philipps-University;

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Citation

Acevedo-Rocha, C. G., Gamble, C. G., Lonsdale, R., Li, A., Nett, N., Hoebenreich, S., et al. (2018). P450-Catalyzed Regio- and Diastereoselective Steroid Hydroxylation: Efficient Directed Evolution Enabled by Mutability Landscaping. ACS Catalysis, 8(4), 3395-3410. doi:10.1021/acscatal.8b00389.


Cite as: https://hdl.handle.net/21.11116/0000-0001-9DD6-D
Abstract
Cytochrome P450 monooxygenases play a crucial role in the biosynthesis of many natural products and in the human metabolism of numerous pharmaceuticals. This has inspired synthetic organic and medicinal chemists to exploit them as catalysts in regio- and stereoselective CH-activating oxidation of structurally simple and complex organic compounds such as steroids. However, levels of regio- and stereoselectivity as well as activity are not routinely high enough for real applications. Protein engineering using rational design or directed evolution has helped in many respects, but simultaneous engineering of multiple catalytic traits such as activity, regioselectivity, and stereoselectivity, while overcoming trade-offs and diminishing returns, remains a challenge. Here we show that the exploitation of information derived from mutability landscapes and molecular dynamics simulations for rationally designing iterative saturation mutagenesis constitutes a viable directed evolution strategy. This combined approach is illustrated by the evolution of P450BM3 mutants which enable nearly perfect regio- and diastereoselective hydroxylation of five different steroids specifically at the C16-position with unusually high activity, while avoiding activity–selectivity trade-offs as well as keeping the screening effort relatively low. The C16 alcohols are of practical interest as components of biologically active glucocorticoids.