A single active site mutation inverts stereoselectivity of 16-hydroxylation of testosterone catalyzed by engineered cytochrome P450 BM3.

Department of Chemistry and Pharmaceutical Sciences, Division of Molecular and Computational Toxicology, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, Netherlands.
ChemBioChem (Impact Factor: 3.06). 03/2012; 13(4):520-3. DOI: 10.1002/cbic.201100750
Source: PubMed

ABSTRACT Inversion of stereoselectivity: screening of a minimal mutant library revealed a cytochrome P450 BM3 variant M01 A82W S72I capable of producing 16 α-OH-testosterone. Remarkably, a single active site mutation S72I in M01 A82W inverted the stereoselectivity of hydroxylation from 16 β to 16 α. Introduction of S72I mutation in another 16 β-OH-selective variant M11 V87I, also resulted in similar inversion of stereoselectivity.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The selective hydroxylation of an unactivated CH bond is a crucial step in the synthesis of fine chemicals such as hydroxylated terpenoids. In the present study, the ability of 40 cytochrome P450 BM3 mutants to perform the regio- and stereoselective hydroxylation of α-ionone has been investigated. Based on their activity and selectivity to produce 3-hydroxy-α-ionone from racemic α-ionone, 6 BM3 mutants were selected. Out of these, 3 mutants (M01 A82W, M11 A82W and M11 V87I) showed high selectivity for trans-3-hydroxy-α-ionone formation while 3 other mutants (M11 L437N, M11 L437S and M11 L437T) formed almost equal amounts of both cis-3-hydroxy- and trans-3-hydroxy-α-ionone. Incubation with individual enantiomers showed that M11 L437N, M11 L437S and M11 L437T exhibited opposite stereoselectivity producing (3S,6S)-hydroxy-α-ionone with the (6S)-enantiomer and (3S,6R)-hydroxy-α-ionone with the (6R)-enantiomer. Thus for the first time, BM3 mutants that can selectively produce diastereomers of 3-hydroxy-α-ionone (>90% de), with high turnover numbers and minimal secondary metabolism, have been identified. Docking studies have been performed to rationalize the basis of the experimentally observed selectivity. In conclusion, engineered P450 BM3s are promising biocatalysts for regio- and stereoselective production of hydroxylated α-ionones for industrial applications.
    Advanced Synthesis & Catalysis 08/2012; 354(11‐12). · 5.54 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cytochrome P450 BM3 mutants are promising biocatalysts for the production of drug metabolites. In the present study, the ability of cytochrome P450 BM3 mutants to produce oxidative metabolites of structurally related NSAIDs meclofenamic acid, mefenamic acid and tolfenamic acid was investigated. A library of engineered P450 BM3 mutants was screened with meclofenamic acid (1) to identify catalytically active and selective mutants. Three mono-hydroxylated metabolites were identified for 1. The hydroxylated products were confirmed by NMR analysis to be 3'-OH-methyl-meclofenamic acid (1a), 5-OH-meclofenamic acid (1b) and 4'-OH-meclofenamic acid (1c) which are human relevant metabolites. P450 BM3 variants containing V87I and V87F mutation showed high selectivity for benzylic and aromatic hydroxylation of 1 respectively. The applicability of these mutants to selectively hydroxylate structurally similar drugs such as mefenamic acid (2) and tolfenamic acid (3) was also investigated. The tested variants showed high total turnover numbers in the order of 4000-6000 and can be used as biocatalysts for preparative scale synthesis. Both 1 and 2 could undergo benzylic and aromatic hydroxylation by the P450 BM3 mutants, whereas 3 was hydroxylated only on aromatic rings. The P450 BM3 variant M11 V87F hydroxylated the aromatic ring at 4' position of all three drugs tested with high regioselectivity. Reference metabolites produced by P450 BM3 mutants allowed the characterisation of activity and regioselectivity of metabolism of all three NSAIDs by thirteen recombinant human P450s. In conclusion, engineered P450 BM3 mutants that are capable of benzylic or aromatic hydroxylation of fenamic acid containing NSAIDs, with high selectivity and turnover numbers have been identified. This shows their potential use as a greener alternative for the generation of drug metabolites.
    Bioorganic & Medicinal Chemistry 06/2014; · 2.95 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Regio- and stereoselective oxidative hydroxylation of achiral or chiral organic compounds mediated by synthetic reagents, catalysts, or enzymes generally leads to the formation of one new chiral center that appears in the respective enantiomeric or diastereomeric alcohols. By contrast, when subjecting appropriate achiral compounds to this type of CH activation, the simultaneous creation of two chiral centers with a defined relative and absolute configuration may result, provided that control of the regio-, diastereo-, and enantioselectivity is ensured. The present study demonstrates that such control is possible by using wild type or mutant forms of the monooxygenase cytochrome P450 BM3 as catalysts in the oxidative hydroxylation of methylcyclohexane and seven other monosubstituted cyclohexane derivatives.
    Angewandte Chemie International Edition 03/2014; · 11.34 Impact Factor