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*

*Corresponding author for this work

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 - Jan 1 2019

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20-Hydroxysteroid Dehydrogenases
Hydrocortisone
Androgens
Bacteria
Oxidoreductases
Steroids
Steroid 17-alpha-Hydroxylase
Clostridium
Gene encoding
Anaerobic Bacteria
Bifidobacterium adolescentis
Protein Stability
Physiology
Probiotics
Enzyme Assays
Chromatography
Metabolism
Glucocorticoids
Genes
Gel Chromatography

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Doden, Heidi L. ; Pollet, Rebecca M. ; Mythen, Sean M. ; Wawrzak, Zdzislaw ; Devendran, Saravanan ; Cann, Isaac ; Koropatkin, Nicole M. ; Ridlon, Jason M. / Structural and biochemical characterization of 20-hydroxysteroid dehydrogenase from Bifidobacterium adolescentis strain L2-32. In: Journal of Biological Chemistry. 2019 ; Vol. 294, No. 32. pp. 12040-12053.
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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 {\AA} 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.",
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Structural and biochemical characterization of 20-hydroxysteroid dehydrogenase from Bifidobacterium adolescentis strain L2-32. / Doden, Heidi L.; Pollet, Rebecca M.; Mythen, Sean M.; Wawrzak, Zdzislaw; Devendran, Saravanan; Cann, Isaac; Koropatkin, Nicole M.; Ridlon, Jason M.

In: Journal of Biological Chemistry, Vol. 294, No. 32, 01.01.2019, p. 12040-12053.

Research output: Contribution to journalArticle

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T1 - Structural and biochemical characterization of 20-hydroxysteroid dehydrogenase from Bifidobacterium adolescentis strain L2-32

AU - Doden, Heidi L.

AU - Pollet, Rebecca M.

AU - Mythen, Sean M.

AU - Wawrzak, Zdzislaw

AU - Devendran, Saravanan

AU - Cann, Isaac

AU - Koropatkin, Nicole M.

AU - Ridlon, Jason M.

PY - 2019/1/1

Y1 - 2019/1/1

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