Metabolism of Oxo-Bile Acids and Characterization of Recombinant 12α-Hydroxysteroid Dehydrogenases from Bile Acid 7α-Dehydroxylating Human Gut Bacteria

Abstract

Bile acids are important cholesterol-derived nutrient signaling hormones, synthesized in the liver, that act as detergents to solubilize dietary lipids. Bile acid 7α-dehydroxylating gut bacteria generate the toxic bile acids deoxycholic acid and lithocholic acid from host bile acids. The ability of these bacteria to remove the 7-hydroxyl group is partially dependent on 7α-hydroxysteroid dehydrogenase (HSDH) activity, which reduces 7-oxo-bile acids generated by other gut bacteria. 3α-HSDH has an important enzymatic activity in the bile acid 7α-dehydroxylation pathway. 12α-HSDH activity has been reported for the low-activity bile acid 7α-dehydroxylating bacterium ; however, this activity has not been reported for high-activity bile acid 7α-dehydroxylating bacteria, such as, , and Here, we demonstrate that these strains express bile acid 12α-HSDH. The recombinant enzymes were characterized from each species and shown to preferentially reduce 12-oxolithocholic acid to deoxycholic acid, with low activity against 12-oxochenodeoxycholic acid and reduced activity when bile acids were conjugated to taurine or glycine. Phylogenetic analysis suggests that 12α-HSDH is widespread among , in the family, and human gut 12α-HSDH activity has been established in the medically important bile acid 7α-dehydroxylating bacteria ,, and Experiments with recombinant 12α-HSDHs from these strains are consistent with culture-based experiments that show a robust preference for 12-oxolithocholic acid over 12-oxochenodeoxycholic acid. Phylogenetic analysis identified novel members of the gut microbiome encoding 12α-HSDH. Future reengineering of 12α-HSDH enzymes to preferentially oxidize cholic acid may provide a means to industrially produce the therapeutic bile acid ursodeoxycholic acid. In addition, a cholic acid-specific 12α-HSDH expressed in the gut may be useful for the reduction in deoxycholic acid concentration, a bile acid implicated in cancers of the gastrointestinal (GI) tract.

Clostridium scindens ATCC 35704: Integration of Nutritional Requirements, the Complete Genome Sequence, and Global Transcriptional Responses to Bile Acids

“…Doubling times for cells grown in BHI, UM, DM, and PO 4 -buffered DM approximated 2.0, 2.3, 3.3, and 7.0 h, respectively. While previous work has shown that CA is 7␣-dehydroxylated to DCA by cells grown in BHI medium (15), we report here CA conversion to DCA by C. scindens ATCC 35704 grown in a defined or minimal medium ( Fig. 1B).…”

“…1C). The bile acid concentration chosen reflects both in vivo physiologically relevant concentrations of bile acids in fecal water (18,19) and concentrations used previously in numerous in vitro studies of C. scindens strains (6,10,15). Separate additions of CA in early and mid-log phase have been previously shown to result in robust induction of the bai regulon (7).…”

“…In contrast, the baiN gene (HDCHB-GLK_03018), previously shown to encode a flavoprotein involved in the reductive arm of the bile acid 7␣-dehydroxylation pathway, was constitutively expressed at an average of 232 Ϯ 63.6 TPM (Data Set S1) (24). The recently reported NADP-dependent 12␣-hydroxysteroid dehydrogenase (HSDH) (HDCHBGLK_00743) characterized from C. scindens ATCC 35704 preferentially converted 12-oxo-LCA to DCA relative to CA metabolites (15). This gene was not differentially regulated by CA or DCA (15).…”

“…The recently reported NADP-dependent 12␣-hydroxysteroid dehydrogenase (HSDH) (HDCHBGLK_00743) characterized from C. scindens ATCC 35704 preferentially converted 12-oxo-LCA to DCA relative to CA metabolites (15). This gene was not differentially regulated by CA or DCA (15). Similarly, a gene encoding NADP-dependent 7␣-HSDH, sharing 98% amino acid identity with NADP-dependent 7␣-HSDH (HDCHBGLK_01258) characterized from C. scindens VPI 12708 (25), was also constitutively expressed at an average of 640 Ϯ 121.5 TPM (Fig.…”

Systematic assessment of secondary bile acid metabolism in gut microbes reveals distinct metabolic capabilities in inflammatory bowel disease

“…To determine how widely genes encoding for bile acid pathways are spread in human gut microbes, we performed a systematic comparative genomic analysis of the bile acid deconjugation and transformation pathway ( Fig. 2), starting with previously characterized enzymes for primary bile acid deconjugation [21] and transformation into secondary bile acids [22][23][24][25]. Of the currently 818 microbial AGORA reconstructions, which include 46 newly reconstructed gut microbes (see the "Materials and methods" for details), only 670 genomes were available at the PubSEED database [26,27].…”