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

Carlos G. Acevedo-Rocha, Charles G. Gamble, Richard Lonsdale, Aitao Li, Nathalie Nett, Sabrina Hoebenreich, Julia B. Lingnau, Cornelia Wirtz, Christophe Fares, Heike Hinrichs, Alfred Deege, Adrian J. Mulholland, Yuval Nov, David Leys, Kirsty J. McLean, Andrew W. Munro, Manfred T. Reetz

Research output: Contribution to journalArticle

34 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)3395-3410
Number of pages16
JournalACS Catalysis
Volume8
Issue number4
Early online date8 Mar 2018
DOIs
Publication statusPublished - 6 Apr 2018
Externally publishedYes

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    Acevedo-Rocha, C. G., Gamble, C. G., Lonsdale, R., Li, A., Nett, N., Hoebenreich, S., Lingnau, J. B., Wirtz, C., Fares, C., Hinrichs, H., Deege, A., Mulholland, A. J., Nov, Y., Leys, D., McLean, K. J., Munro, A. W., & Reetz, M. T. (2018). P450-Catalyzed Regio- and Diastereoselective Steroid Hydroxylation: Efficient Directed Evolution Enabled by Mutability Landscaping. ACS Catalysis, 8(4), 3395-3410. https://doi.org/10.1021/acscatal.8b00389