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

• Published in: Nature chemistry 2011-09, Vol.3 (9), p.738-743
• Main Authors: Kille, Sabrina, Zilly, Felipe E., Acevedo, Juan P., Reetz, Manfred T.
• Format: Article
• Language: English
• Subjects: 631/61/191/1908; 639/638/45/607; 639/638/549; 639/638/563/981; Activation; Alcohol; Alcohols; Analytical Chemistry; Bacillus megaterium - enzymology; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biocatalysis; Biochemistry; Catalytic Domain; Chemistry; Chemistry/Food Science; Cytochrome; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450; Cytochromes P450; Directed evolution; Directed Molecular Evolution - methods; Enzymes; Evolution; Hydroxylation; Inorganic Chemistry; Kinetics; Models, Molecular; Molecular dynamics; Mutagenesis; Mutants; NADP - metabolism; NADPH-Ferrihemoprotein Reductase - chemistry; NADPH-Ferrihemoprotein Reductase - genetics; NADPH-Ferrihemoprotein Reductase - metabolism; Organic Chemistry; Oxidation; Oxidation-Reduction; Physical Chemistry; Saturation mutagenesis; Stereoisomerism; Stereoselectivity; Steroid hormones; Steroids; Substrate Specificity; Substrates; Testosterone; Testosterone - chemistry; Testosterone - metabolism
• ISSN: 1755-4330; 1755-4349
• DOI: 10.1038/nchem.1113

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. Selective reaction of one C–H bond among many in complex organic molecules is a grand challenge for organic chemistry. Here, starting from an enzyme that oxidizes two positions in a steroid without bias, laboratory evolution is used to prepare mutants that can regio- and stereoselectively oxidize either position.