Structural and biochemical characterization of 20β-hydroxysteroid dehydrogenase from Bifidobacterium adolescentis strain L2-32

• Published in: The Journal of biological chemistry 2019-08, Vol.294 (32), p.12040-12053
• Main Authors: Doden, Heidi L., Pollet, Rebecca M., Mythen, Sean M., Wawrzak, Zdzislaw, Devendran, Saravanan, Cann, Isaac, Koropatkin, Nicole M., Ridlon, Jason M.
• Format: Article
• Language: English
• Subjects: 20β-hydroxysteroid dehydrogenase; androgen; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; BASIC BIOLOGICAL SCIENCES; Bifidobacteria; Bifidobacterium adolescentis - enzymology; C-20 reduction; cortisol; Crystallography, X-Ray; Enzymology; glucocorticoid; Hydrocortisone - chemistry; Hydrocortisone - metabolism; Hydroxysteroid Dehydrogenases - chemistry; Hydroxysteroid Dehydrogenases - genetics; Hydroxysteroid Dehydrogenases - metabolism; INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Kinetics; microbiome; Mutagenesis, Site-Directed; NAD - chemistry; NAD - metabolism; oxidation–reduction (redox); probiotic; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Steroid-17,20-desmolase; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.009390

Anaerobic bacteria inhabiting the human gastrointestinal tract have evolved various enzymes that modify host-derived steroids. The bacterial steroid-17,20-desmolase pathway cleaves the cortisol side chain, forming pro-androgens predicted to impact host physiology. Bacterial 20β-hydroxysteroid dehydrogenase (20β-HSDH) regulates cortisol side-chain cleavage by reducing the C-20 carboxyl group on cortisol, yielding 20β-dihydrocortisol. Recently, the gene encoding 20β-HSDH in Butyricicoccus desmolans ATCC 43058 was reported, and a nonredundant protein search yielded a candidate 20β-HSDH gene in Bifidobacterium adolescentis strain L2-32. B. adolescentis 20β-HSDH could regulate cortisol side-chain cleavage by limiting pro-androgen formation in bacteria such as Clostridium scindens and 21-dehydroxylation by Eggerthella lenta. Here, the putative B. adolescentis 20β-HSDH was cloned, overexpressed, and purified. 20β-HSDH activity was confirmed through whole-cell and pure enzymatic assays, and it is specific for cortisol. Next, we solved the structures of recombinant 20β-HSDH in both the apo- and holo-forms at 2.0–2.2 Å resolutions, revealing close overlap except for rearrangements near the active site. Interestingly, the structures contain a large, flexible N-terminal region that was investigated by gel-filtration chromatography and CD spectroscopy. This extended N terminus is important for protein stability because deletions of varying lengths caused structural changes and reduced enzymatic activity. A nonconserved extended N terminus was also observed in several short-chain dehydrogenase/reductase family members. B. adolescentis strains capable of 20β-HSDH activity could alter glucocorticoid metabolism in the gut and thereby serve as potential probiotics for the management of androgen-dependent diseases.