Steroid metabolism in the human gut by microbial hydroxysteroid dehydrogenases

Bile acids and cortisol are steroid hormones derived from cholesterol that are important signaling molecules in the human body. Bile acids are essential for nutrient solubilization and absorption in the gut while cortisol is involved in stress response and metabolism throughout the body. The gut microbiome has evolved a network of enzymes that modify steroids produced by the host. One such enzyme activity is the oxidoreduction of steroid hydroxyl groups by hydroxysteroid dehydrogenases (HSDHs). Pairs of HSDHs can reversibly epimerize α-hydroxyl groups through an oxo-intermediate to β-hydroxyl groups. These small modifications of steroid structure often greatly influence their physicochemical properties, leading to changes in toxicity, solubility, and their ability to activate or inhibit host receptors. In this work, two types of bile acid HSDHs were explored: 12α-HSDH, which reversibly converts the C-12 position hydroxyl from the α-orientation to an oxo-group, and 12β-HSDH, which completes the epimerization by converting the C-12 oxo-group to the β-orientation. Three 12α-HSDHs were identified in the human gut microbes Clostridium scindens ATCC 35704, C. hylemonae DSM 15053, and Peptacetobacter hiranonis DSM 13275. After cloning and overexpression of these enzymes in Escherichia coli, biochemical analysis revealed a preference for the oxo-intermediate, 12-oxolithocholic (12-oxoLCA), over the 12α-hydroxy substrate, deoxycholic acid (DCA). Phylogenetic analysis suggests 12α-HSDH is widespread across Firmicutes and Actinobacteria. The first gene encoding bile acid 12β-HSDH, converting 12-oxoLCA to epiDCA, was discovered following a screen of 6 candidate enzymes from C. paraputrificum ATCC 25780. A phylogenetic analysis of 12β-HSDH led to the identification of two additional validated bile acid 12β-HSDHs in Eisenbergiella sp. OF01-20 and Olsenella sp. GAM18. Comparison of the 12α- and 12β-HSDH phylogenies revealed two shared organisms between them, Collinsella tanakaei YIT 12063 and Collinsella stercoris DSM 13279. Although not yet confirmed in culture, these are the first strains identified with potential bile acid C-12 epimerizing activity. Additionally, two HSDHs metabolizing cortisol were characterized biochemically and structurally: 20β-HSDH, which interconverts cortisol and 20β-dihydrocortisol, and 20α-HSDH, which reversibly biotransforms cortisol to 20α-dihydrocortisol. A 20β-HSDH from Bifidobacterium adolescentis strain L2-32 was cloned, overexpressed and purified. The apo-form without ligands bound and the binary complex with cofactor bound were crystallized. A large, flexible N-terminal domain within the structures was investigated further by gel filtration chromatography and circular dichroism spectroscopy. Deletions of the extended N-terminus caused a loss of activity and structural changes, suggesting the N-terminus is critical for protein stability. Additionally, a 20α-HSDH from C. scindens ATCC 35704 was crystallized and its enzymatic mechanism predicted by hybrid quantum mechanics/molecular mechanics (QM/MM) simulations. In order to test the reaction mechanism, substrate-binding and catalytic residues were validated by site-directed mutagenesis and isothermal titration calorimetry. Microbial HSDHs are predicted regulators of steroid pathways implicated in colorectal cancer, liver cancer, castration-resistant prostate cancer and polycystic ovary syndrome. Due to their regulatory potential, microbial HSDHs may have promise as therapeutics or druggable targets in the future. Thus, the characterization of new bile acid 12α/β-HSDH and cortisol 20α/β-HSDH activities will serve as the foundation for future mechanistic studies on the role of HSDHs in various disease states