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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.

Author information

1
Department of Life Science and Biochemical Engineering, Sunmoon University, Asansi, Korea.
2
Unit of Polar Genomics, Korea Polar Research Institute, Incheon, Korea.
3
Department of Polar Sciences, University of Science and Technology, Incheon, Korea.
4
Genome-based BioIT Convergence Institute, Asansi, Korea.
5
Department of Pharmaceutical Engineering and Biotechnology, Sunmoon University, Asansi, Korea.

Abstract

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.

KEYWORDS:

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

PMID:
30552795
DOI:
10.1111/febs.14729

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