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

Abstract

Abstract:The selective hydroxylation of an unactivated C À 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 a-ionone has been investigated. Based on their activity and selectivity to produce 3-hydroxy-a-ionone from racemic a-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-a-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-a-ionone. Incubation with individual enantiomers showed that M11 L437N, M11 L437S and M11 L437T exhibited opposite stereoselectivity producing (3S,6S)-hydroxya-ionone with the (6S)-enantiomer and (3S,6R)-hydroxy-a-ionone with the (6R)-enantiomer. Thus for the first time, BM3 mutants that can selectively produce diastereomers of 3-hydroxy-a-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 a-ionones for industrial applications.

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

“…22,25 In order to rationalize the observed ring preference for hydroxylation, in silico docking studies were performed with BM3 mutant M11 and 2 as a model, using the GOLD (version 5.2) software 35 and the docking settings as previously described. 36 Docking of 2 into M11 predominantly shows the occurrence of binding poses in which 2 is docked with its dimethyl phenyl ring toward the heme group, with either 3'-methyl or C-4' in closest proximity of the heme iron center (see Figure 5, panels A and B, respectively). These binding orientations are stabilized by formation of a hydrogen bond between the 2 carboxyl group and the side-chain hydroxyl group of Ser72.…”

Selective whole-cell biosynthesis of the designer drug metabolites 15- or 16-betahydroxynorethisterone by engineered Cytochrome P450 BM3 mutants

“…Also within our own lab, isolated CYP BM3 mutants have previously been successfully applied for biosynthesis of drug metabolites on a preparative scale [32][33][34][35][36]39,47,48]. During our in vitro experiment, bioconversion was monitored in time (see Fig.…”

“…To overcome this limitation, various protein engineering strategies have been successfully applied to generate CYP BM3 mutants that can be used for the production of both human relevant and CYP BM3 unique metabolites [2,3,26]. Over the years, a diverse library of CYP BM3 mutants has been constructed within our group by a combination of random and site-directed mutagenesis [27][28][29][30][31][32][33][34]. It has been shown that these mutants metabolize a wide range of substrates, including marketed drugs [28,30,35,36] and steroids [29,32,34], with varying catalytic activities and regio-and stereoselectivities.…”

“…Detailed information about the mutations present in the different mutants is tabulated in the Supporting information, Table S1. For 44 of the CYP BM3 mutants construction has been described previously [28][29][30][31][32][33][34]. In total 22 novel mutants were made by site-directed mutagenesis using the appropriate oligonucleotides and templates (see the Supporting Information, Table S1).…”

Structure‐Based Methods for Predicting the Sites and Products of Metabolism

“…Various drug metabolizing mutants of cytochrome CYP102A1 from Bacillus megaterium (CYP BM3) have been described, and from the onset, computational tools were integrated in the rationalization and design of novel mutants [49,71,72]. Two mutants of CYPBM3 were experimentally shown to catalyze the stereospecific hydroxylation of α-ionones [73]. By using efficient free energy calculations, the relative binding free energies of the substrates and the products could be determined in both mutants (see Figure 10.5).…”