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

Heidi L. Doden, Rebecca M. Pollet, Sean M. Mythen, Zdzislaw Wawrzak, Saravanan Devendran, Isaac Cann, Nicole M. Koropatkin, Jason M. Ridlon

Research output: Contribution to journalArticle

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 20-hydroxysteroid dehydrogenase (20-HSDH) regulates cortisol side-chain cleavage by reducing the C-20 carboxyl group on cortisol, yielding 20-dihydrocortisol. Recently, the gene encoding 20-HSDH in Butyricicoccus desmolans ATCC 43058 was reported, and a nonredundant protein search yielded a candidate 20-HSDH gene in Bifidobacterium adolescentis strain L2-32. B. adolescentis 20-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 20-HSDH was cloned, overexpressed, and purified. 20-HSDH activity was confirmed through whole-cell and pure enzymatic assays, and it is specific for cortisol. Next, we solved the structures of recombinant 20-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 20-HSDH activity could alter glucocorticoid metabolism in the gut and thereby serve as potential probiotics for the management of androgen-dependent diseases.

Original languageEnglish (US)
Pages (from-to)12040-12053
Number of pages14
JournalJournal of Biological Chemistry
Volume294
Issue number32
DOIs
StatePublished - Aug 9 2019

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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