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

Abstract

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.

Keywords: 20β-hydroxysteroid dehydrogenase; Bifidobacteria; C-20 reduction; Steroid-17,20-desmolase; androgen; cortisol; glucocorticoid; microbiome; oxidation–reduction (redox); probiotic.

Figure 1.

Cortisol-metabolizing reactions by B. adolescentis, C. scindens, B. desmolans, P. lymphophilum, and E. lenta. Boxed reaction shows 20β-HSDH oxidizes the coenzyme NADH to transform cortisol into 20β-dihydrocortisol. Cortisol + NADH ⇌ 20β-dihydrocortisol + NAD⁺. This reaction reduces the double-bonded oxygen at C-20 (highlighted in red) on cortisol and is fully reversible.

Figure 2.

Whole-cell assays of B. adolescentis strain L2-32, B. desmolans ATCC 43058, and C. scindens ATCC 35704. A, B. adolescentis completely converts cortisol into 20β-dihydrocortisol. 20α-Dihydrocortisol is not metabolized. In a highly reductive environment and without pressure from a steroid-17,20-desmolase, B. adolescentis does not convert 20β-dihydrocortisol into cortisol. B, B. desmolans completely converts cortisol into 11β-OHAD and 20β-dihydrocortisol into 11β-OHAD. 20α-Dihydrocortisol is not metabolized. C, C. scindens completely converts cortisol into 11β-OHAD and 20α-dihydrocortisol into 11β-OHAD. 20β-Dihydrocortisol is not metabolized. Standards are reused in each panel for ease of comparison to reaction products above.

Figure 3.

Purified 20β-HSDH from B. adolescentis, strain L2-32. A, SDS-polyacrylamide gel of crude and purified 20β-HSDH showing a subunit size of 32 ± 0.12 kDa. Lane M, molecular mass markers. B, native molecular size analysis of purified 20β-HSDH via gel-filtration chromatography.

Figure 4.

pH optimum and Michaelis-Menten saturation curve of WT 20β-HSDH. A, pH optimization of 20β-HSDH in the reductive (blue) and oxidative (red) direction. B, saturation curve showing the reduction of cortisol by WT 20β-HSDH.

Figure 5.

Structural characterization of 20β-HSDH. A, apo structure of 20β-HSDH showing the SDR-characteristic Rossmann fold consisting of seven central β-strands (β3–β2–β1–β4–β5–β6–β7) flanked by six α-helices. Right panel depicts the extended N terminus with no electron density before residue 17 and truncation sites. B, proposed tetramer of 20β-HSDH based on crystallographic arrangement. C, NADH-binding pocket showing five side chains and three peptidyl backbone interactions. D, surface representation of the NADH-binding pocket highlighting residues that make van der Waals contacts to NADH (yellow). The Gly-rich region is highlighted in pink and also makes van der Waals contacts to NADH.

Figure 6.

CD analysis and relative activity of WT and truncated 20β-HSDH. A, relative activity based on spectrophotometric assay. B, thermal stability of WT, 17-, and 21-truncated 20β-HSDH by temperature-dependent CD. C, percentage secondary structure calculated with DichroWeb according to D and CD spectra of WT and truncated proteins.