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FEBS J. 2019 May;286(9):1683-1699. doi: 10.1111/febs.14729. Epub 2018 Dec 31.

Characterization of two steroid hydroxylases from different Streptomyces spp. and their ligand-bound and -unbound crystal structures.

Dangi B1, Lee CW2,3, Kim KH1, Park SH2, Yu EJ1, Jeong CS2,3, Park H2,3, Lee JH2,3, Oh TJ1,4,5.

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Department of Life Science and Biochemical Engineering, Sunmoon University, Asansi, Korea.
Unit of Polar Genomics, Korea Polar Research Institute, Incheon, Korea.
Department of Polar Sciences, University of Science and Technology, Incheon, Korea.
Genome-based BioIT Convergence Institute, Asansi, Korea.
Department of Pharmaceutical Engineering and Biotechnology, Sunmoon University, Asansi, Korea.


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.


Streptomyces ; X-ray crystallography; crystal structure; cytochrome P450; steroid hydroxylase


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