In vivo and In vitro hydroxylation of Cineole and Camphor by Cytochromes P450CYP101A1, CYP101B1 and N242A CYP176A1

In this study three bacterial P450s, P450cam (CYP101A1), CYP101B1 and the mutant N242A-P450cin (N242A-CYP176A1), were used to produce a range of products from the oxidation of the monoterpenes (1R)- and (1S)-camphor and 1,8-cineole. We demonstrate that both in vitro and in vivo catalytic turnover with these P450s can produce a complement of up to seven hydroxycamphors and seven hydroxycineoles, in addition to compounds produced from further oxidation. The CYP101B1 whole cell catalytic system was found to produce 300–600mg/L of culture of oxidation products that could be easily separated chromatographically. The CYP101B1 in vitro oxidation of 1,8-cineole primarily produced (1S)-5α-hydroxycineole, which was 78% of the total product formed. However, the amount of (1S)-5α-hydroxycineole was reduced to 42% of the total products when isolated from the CYP101B1 whole cell system. (1S)-6α-Hydroxycineole (96% ee) could be isolated from a whole cell catalytic turnover of 1,8-cineole by N242A-P450cin in a yield of 46mg/L (98% of the total product). However, the amount of product isolated ((1R)-5-endo-hydroxycamphor, 75% of the total products) from the whole cell catalytic oxidation of (1R)-camphor with N242A-P450cin was much lower (6mg/L) due to the inefficient use of reducing equivalents (3.5±0.5%) for substrate oxidation. These compounds will assist in the identification of specific structures in mechanistic investigations and structure elucidation, but further optimisation is r...
Source: Journal of Molecular Catalysis B: Enzymatic - Category: Biochemistry Source Type: research