Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution

Sabrina Kille, Felipe E. Zilly, Juan P. Acevedo, Manfred T. Reetz*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

241 Scopus citations

Abstract

A current challenge in synthetic organic chemistry is the development of methods that allow the regio- and stereoselective oxidative C - H activation of natural or synthetic compounds with formation of the corresponding alcohols. Cytochrome P450 enzymes enable C - H activation at non-activated positions, but the simultaneous control of both regio- and stereoselectivity is problematic. Here, we demonstrate that directed evolution using iterative saturation mutagenesis provides a means to solve synthetic problems of this kind. Using P450 BM3(F87A) as the starting enzyme and testosterone as the substrate, which results in a 1:1 mixture of the 2β- and 15β-alcohols, mutants were obtained that are 96 - 97% selective for either of the two regioisomers, each with complete diastereoselectivity. The mutants can be used for selective oxidative hydroxylation of other steroids without performing additional mutagenesis experiments. Molecular dynamics simulations and docking experiments shed light on the origin of regio- and stereoselectivity.

Original languageEnglish
Pages (from-to)738-743
Number of pages6
JournalNature Chemistry
Volume3
Issue number9
DOIs
StatePublished - Sep 2011
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by the Fonds der Chemischen Industrie. The authors thank F. Schulz, S. Bastian, J. Drone and D. Bougioukou for discussions, H. Hinrichs and A. Deege for HPLC analyses and C. Farès for NMR analyses.

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