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

Carlos G. Acevedo-Rocha, Charles 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 Mclean, Andrew Munro, Manfred T Reetz

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


    • Directed evolution
    • Cytochrome P450 monooxygenase
    • Regioselectivity
    • Stereoselectivity
    • mutability landscapes
    • iterative saturation mutagenesis
    • Steroids
    • C-H activation

    Research Beacons, Institutes and Platforms

    • Biotechnology
    • Manchester Institute of Biotechnology


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