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

• Published in: The FEBS journal 2019-05, Vol.286 (9), p.1683-1699
• Main Authors: Dangi, Bikash, Lee, Chang Woo, Kim, Ki‐Hwa, Park, Sun‐Ha, Yu, Eun‐Ji, Jeong, Chang‐Sook, Park, Hyun, Lee, Jun Hyuck, Oh, Tae‐Jin
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Androstenedione - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Biocatalysts; Catalytic Domain; Chromatography, High Pressure Liquid; Conserved Sequence; Crystal structure; Crystallography, X-Ray; Cytochrome; Cytochrome P450; Cytochromes P450; Enzymes; Heme; Hydroxyl groups; Hydroxylation; Ligands; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Organic chemistry; Progesterone - metabolism; Protein Binding; Protein Conformation; Protein engineering; Proteins; Recombinant Proteins - chemistry; Regioselectivity; Sequence Alignment; Sequence Homology, Amino Acid; steroid hydroxylase; Steroid Hydroxylases - chemistry; Steroid Hydroxylases - metabolism; Steroids; Streptomyces; Streptomyces - enzymology; Substrates; Testosterone; Testosterone - metabolism; X‐ray crystallography
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.14729

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. This study characterizes two heme‐containing bacterial cytochrome P450 (CYP) enzymes from Streptomyces spp. Crystal structures of the ligand‐unbound (StCYP154C4‐1) form and the testosterone‐bound (StCYP154C4‐2) form are provided, revealing different active site residue compositions that might explain variations in the regiospecificity of steroid substrates among the different CYP154 family members. Hydroxylation at a C‐2 position of testosterone occurs via a mechanism that differs from that of other members of the CYP154 family.