Completion of the gut microbial epi-bile acid pathway

Abstract

Bile acids are detergent molecules that solubilize dietary lipids and lipid-soluble vitamins. Humans synthesize bile acids with α-orientation hydroxyl groups which can be biotransformed by gut microbiota to toxic, hydrophobic bile acids, such as deoxycholic acid (DCA). Gut microbiota are also capable of converting hydroxyl groups from the α-orientation through an oxo-intermediate to the β-orientation, resulting in more hydrophilic and less toxic bile acids. This interconversion is catalyzed by regio- (C-3 vs. C-7) and stereospecific (α vs. β) hydroxysteroid dehydrogenases (HSDHs). Recently, multiple human gut clostridia have been reported to encode 12α-HSDH, which interconverts DCA and 12-oxolithocholic acid (12-oxoLCA). Bile acid 12β-HSDH activity completes the epimerization of DCA by converting 12-oxoLCA to the 12β-bile acid known as epiDCA. While 12β-HSDH activity has been shown in cell extracts of Clostridium paraputrificum, the gene has not yet been reported. In order to identify the first gene encoding this activity, 6 candidate oxidoreductase genes from C. paraputrificum ATCC 25780 were cloned, overexpressed, purified, and screened for activity with 12-oxoLCA and epiDCA. LC-MS analysis was performed on reaction products from the enzyme encoded by DR024_RS09610, confirming the first 12β-HSDH gene discovered. The enzyme was more specific for bile acids lacking a 7-hydroxyl group than cholic acid derivatives containing a 7-hydroxyl. Phylogenetic analysis revealed previously unknown diversity for bile acid 12β-HSDH by experimentally validating two additional 12β-HSDHs within the tree from Eisenbergiella sp. OF01-20 and Olsenella sp. GAM18.

Microbial Hydroxysteroid Dehydrogenases: From Alpha to Omega

“…This causes great difficulty in predicting substrate specificities by amino-acid homology search alone. HSDHs within the SDR superfamily include but are not limited to host 11β-HSD and 17β-HSD [5], and various microbial BA 12α-HSDHs [23], 12β-HSDH [24], 3α/β-HSDHs [17], and glucocorticoid 20β-HSDH [15].…”

“…Compared to the iso-and urso-BA pathways, the least is known about the epi-BA pathway. While multiple 12α-HSDHs have been characterized [18,23,103,116,129,130], BA 12β-HSDH was only studied in cell extracts until the discovery of the first gene encoding this activity by our lab [24,131,132]. 12-Oxolithocholic acid (12-oxoLCA; 3α-hydroxy,12oxo), the product of 12α-HSDH oxidation of DCA, is often one of the most abundant oxo-BAs found in human feces, at concentrations of about one half DCA in some studies [81,133,134].…”

“…12α-HSDHs generally have higher activity with free and dihydroxy-BAs than conjugated or trihydroxy-BAs [18,23,129]. The only gut microbial BA 12β-HSDH characterized to date, from Clostridium paraputrificum ATCC 25780, has affinity for NADP(H) and greater activity with dihydroxy-BAs [24,132]. Two additional 12β-HSDHs have been shown to react with 12-oxoLCA and epiDCA with NADP(H) as co-substrate, although their substrate specificities have not been fully characterized [24].…”

“…The only gut microbial BA 12β-HSDH characterized to date, from Clostridium paraputrificum ATCC 25780, has affinity for NADP(H) and greater activity with dihydroxy-BAs [24,132]. Two additional 12β-HSDHs have been shown to react with 12-oxoLCA and epiDCA with NADP(H) as co-substrate, although their substrate specificities have not been fully characterized [24]. Interestingly, 12β-HSDH activity recognizing side-chain cleaved steroids derived from BAs has been observed in multiple environmental microorganisms.…”

“…Epi-BAs are understudied compared to urso-and iso-BAs. Thus, their toxicity relative to secondary BAs is untested, although epiDCA and 12-oxoLCA are less hydrophobic than DCA according to LC-MS [24]. It is possible that isomerization of primary BAs to iso-or epi-BAs may impede formation of secondary BAs if they cannot be recognized by 7-dehydroxylation pathway enzymes.…”