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

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.

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Acevedo-Rocha, Carlos G.1, 2, Author           
Gamble, Charles G.3, Author
Lonsdale, Richard1, 2, 4, Author           
Li, Aitao1, 2, 5, Author           
Nett, Nathalie2, Author
Hoebenreich, Sabrina2, Author
Lingnau, Julia B.6, Author           
Wirtz, Cornelia6, Author           
Fares, Christophe6, Author           
Hinrichs, Heike7, Author           
Deege, Alfred7, Author           
Mulholland, Adrian J.4, Author
Nov, Yuval8, Author
Leys, David3, Author
McLean, Kirsty J.3, Author
Munro, Andrew W.3, Author
Reetz, Manfred T.1, 2, Author           
1Research Department Reetz, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445588              
2Department of Chemistry, Philipps-University, Hans-Meerwein-Strasse 4, 35032 Marburg, Germany, ou_persistent22              
3Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester M1 7DN, U.K., ou_persistent22              
4Centre for Computational Chemistry, School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, U.K., ou_persistent22              
5Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, 368 Youyi Road, Wuchang Wuhan 430062, China, ou_persistent22              
6Service Department Farès (NMR), Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445623              
7Service Department Schulze (GC, HPLC), Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_persistent22              
8Department of Statistics, University of Haifa, Haifa 31905, Israel, ou_persistent22              


Free keywords: directed evolution; cytochrome P450 monooxygenase; regioselectivity; stereoselectivity; mutability landscapes; iterative saturation mutagenesis; steroids; C-H activiation
 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.


Language(s): eng - English
 Dates: 2018-01-292018-03-082018-04-06
 Publication Status: Published online
 Pages: 16
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/acscatal.8b00389
 Degree: -



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Title: ACS Catalysis
  Abbreviation : ACS Catal.
Source Genre: Journal
Publ. Info: Washington, DC : ACS
Pages: - Volume / Issue: 8 (4) Sequence Number: - Start / End Page: 3395 - 3410 Identifier: Other: 2155-5435
CoNE: https://pure.mpg.de/cone/journals/resource/2155-5435