Regio- and Stereoselective Hydroxylation of Optically Active α-Ionone Enantiomers by Engineered Cytochrome P450 BM3 Mutants

The selective hydroxylation of an unactivated C-H 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 alpha-ionone has been investigated. Based on their activity and selectivity to produce 3-hydroxy-alpha-ionone from racemic alpha-ionone, 6 BM3 mutants were selected. Out of these, 3 mutants (M01A82W, M11A82W and M11V87I) showed high selectivity for trans-3-hydroxy-alpha-ionone formation while 3 other mutants (M11L437N, M11L437S and M11L437T) formed almost equal amounts of both cis-3-hydroxy- and trans-3-hydroxy-alpha-ionone. Incubation with individual enantiomers showed that M11L437N, M11L437S and M11L437T exhibited opposite stereoselectivity producing (3S,6S)-hydroxy-alpha-ionone with the (6S)-enantiomer and (3S,6R)-hydroxy-alpha-ionone with the (6R)-enantiomer. Thus for the first time, BM3 mutants that can selectively produce diastereomers of 3-hydroxy-alpha-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 alpha-ionones for industrial applications.