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.

Glucosylation of the 21-hydroxyl group of glucocorticoid changes its solubility into hydrophilicity from hydrophobicity and, as with glucocorticoid glucuronides as a moving object in vivo, it is conceivable that it exhibits the same behavior. Therefore, glucosylation to the 21-hydroxyl group while maintaining the 11β-hydroxyl group is particularly important, and glucosylation of corticosterone was confirmed by high-resolution mass spectrometry and 1D (1H and 13C) and 2D (COSY, ROESY, HSQC-DEPT and HMBC) NMR. Moreover, the difference in bioactivity between corticosterone and corticosterone 21-glucoside was investigated in vitro. Corticosterone 21-glucoside showed greater neuroprotective effects against H2O2-induced cell death and reactive oxygen species (ROS) compared with corticosterone. These results for the first time demonstrate that bioconversion of corticosterone through the region-selective glucosylation of a novel compound can present structural potential for developing new neuroprotective agents.

The application of steroids has steadily increased thanks to their therapeutic effects. However, alternatives are required due their severe side effects; thus, studies on the activities of steroid derivatives are underway. Sugar derivatives of nandrolone, which is used to treat breast cancer, as well as cortisone and prednisone, which reduce inflammation, pain, and edema, are unknown. We linked O-glucose to nandrolone and testosterone using UDP-glucosyltransferase (UGT-1) and, then, tested their bioactivities in vitro. Analysis by NMR showed that the derivatives were 17β-nandrolone β-D-glucose and 17β-testosterone β-D-glucose, respectively. The viability was higher and cytotoxicity was evident in PC12 cells incubated with rotenone and, testosterone derivatives, compared to the controls. SH-SY5Y cells incubated with H 2 O 2 and nandrolone derivatives remained viable and cytotoxicity was attenuated. Both derivatives enhanced neuronal protective effects and increased the amounts of cellular ATP.

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