Premium
Characterization of two steroid hydroxylases from different Streptomyces spp. and their ligand‐bound and ‐unbound crystal structures
Author(s) -
Dangi Bikash,
Lee Chang Woo,
Kim KiHwa,
Park SunHa,
Yu EunJi,
Jeong ChangSook,
Park Hyun,
Lee Jun Hyuck,
Oh TaeJin
Publication year - 2019
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14729
Subject(s) - hydroxylation , steroid , stereochemistry , cytochrome p450 , streptomyces , ligand (biochemistry) , enzyme , chemistry , cofactor , biochemistry , heme , cytochrome , biology , receptor , bacteria , genetics , hormone
Bacterial cytochrome P450 ( CYP ) enzymes are involved in the hydroxylation of various endogenous substrates while using a heme molecule as a cofactor. CYP s 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 CYP 154C4 proteins from Streptomyces sp. W2061 ( St CYP 154C4‐1) and Streptomyces sp. ATCC 11861 ( St CYP 154C4‐2). Activity assays showed that both St CYP 154C4‐1 and St CYP 154C4‐2 can produce 2′‐hydroxylated testosterone, which differs from the activity of a previously described Nf CYP 154C5 from Nocardia farcinica in terms of its 16α‐hydroxylation of testosterone. To better understand the molecular basis of the regioselectivity of these two CYP 154C4 proteins, crystal structures of the ligand‐unbound form of St CYP 154C4‐1 and the testosterone‐bound form of St CYP 154C4‐2 were determined. Comparison with the previously determined Nf CYP 154C5 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 CYP s exhibiting different regiospecificity.