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 2 alpha-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 angstrom resolution. The structure has a closed conformation, and the substrate binding pocket is narrow, which can explain the strict substrate specificity of the enzyme. (C) 2019 Elsevier Inc. All rights reserved.