Regio- and stereoselective hydroxylation of testosterone by a novel cytochrome P450 154C2 from Streptomyces avermitilis

Cytochrome P450 enzymes (P450 or CYP) are some of the most versatile biocatalysts , and offer advantages for oxidizing unreactive C–H bonds in mild conditions. In this study, we identified a novel cytochrome P450 154C2 from Streptomyces avermitilis and characterized its function in 2α-hydroxylation of testosterone with regio- and stereoselectivity . To investigate the efficiency of electron transfer , we conducted biotransformation using two different P450 redox partners—RhFRED (RhF reductase domain) from Rhodococcus sp. and Pdx (putidaredoxin)/Pdr (putidaredoxin reductase) from Pseudomonas putida and revealed that RhFRED was more effective than Pdx/Pdr, especially in vivo . The K m and k cat values for testosterone were estimated to be 0.16 ± 0.05 mM and 0.13 ± 0.02 min −1 , and k cat / K m was 0.81 min −1  mM −1 . We also determined the crystal structure of the substrate-free form of CYP154C2 at 1.5 Å resolution. The structure has a closed conformation, and the substrate binding pocket is narrow, which can explain the strict substrate specificity of the enzyme .