CYP154C8 from Streptomyces sp. has been identified as a new cytochrome P450 with substrate flexibility towards different sets of steroids. In vitro treatment of these steroids with CYP154C8 revealed interesting product formation patterns with the same group of steroids. NMR study revealed the major product of corticosterone to be hydroxylated at the C21 position, whereas progesterone, androstenedione, testosterone, and 11-ketoprogesterone were exclusively hydroxylated at the 16α position. However, the 16α-hydroxylated product of progesterone was further hydroxylated to yield dihydroxylated products. 16-hydroxyprogesterone was hydroxylated at two positions to yield dihydroxylated products: 2α,16α-dihydroxyprogesterone and 6β,16α-dihydroxyprogesterone. To the best of our knowledge, this is the first report of generation of such products through enzymatic hydroxylation by a CYP450. In view of the importance of modified steroids as pharmaceutical components, CYP154C8 has immense potential for utilization in bioproduction of hydroxylated derivative compounds to be directly employed for pharmaceutical applications.

Bacterial cytochrome P450 (CYP) enzymes are involved in the hydroxylation of various endogenous substrates while using a heme molecule as a cofactor. CYPs have gained biotechnological interest as useful biocatalysts capable of altering chemical structures by adding a hydroxyl group in a regiospecific manner. Here, we identified, purified, and characterized two CYP154C4 proteins from Streptomyces sp. W2061 (StCYP154C4‐1) and Streptomyces sp. ATCC 11861 (StCYP154C4‐2). Activity assays showed that both StCYP154C4‐1 and StCYP154C4‐2 can produce 2′‐hydroxylated testosterone, which differs from the activity of a previously described NfCYP154C5 from Nocardia farcinica in terms of its 16α‐hydroxylation of testosterone. To better understand the molecular basis of the regioselectivity of these two CYP154C4 proteins, crystal structures of the ligand‐unbound form of StCYP154C4‐1 and the testosterone‐bound form of StCYP154C4‐2 were determined. Comparison with the previously determined NfCYP154C5 structure revealed differences in the substrate‐binding residues, suggesting a likely explanation for the different patterns of testosterone hydroxylation, despite the high sequence similarities between the enzymes (54% identity). These findings provide valuable insights that will enable protein engineering for the development of artificial steroid‐related CYPs exhibiting different regiospecificity.

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The bacterial CYP105 family is involved in secondary metabolite biosynthetic pathways and plays essential roles in the biotransformation of xenobiotics. This study investigates the newly identified H2O2-mediated CYP105D18 from Streptomyces laurentii as the first bacterial CYP for N-oxidation. The catalytic efficiency of CYP105D18 for papaverine N-oxidation was 1.43 s−1 µM −1. The heme oxidation rate (k) was low (<0.3 min−1) in the presence of 200 mM H2O2. This high H2O2 tolerance capacity of CYP105D18 led to higher turnover prior to heme oxidation. Additionally, the high-resolution papaverine complexed structure and substrate-free structure of CYP105D18 were determined. Structural analysis and activity assay results revealed that CYP105D18 had a strong substrate preference for papaverine because of its bendable structure. These findings establish a basis for biotechnological applications of CYP105D18 in the pharmaceutical and medicinal industries.

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