Publications

• Cloning and sequencing of a bile acid-inducible operon from Eubacterium sp. strain VPI 12708.

  Mallonee D.H., White W.B., Hylemon P.B.

  Two bile acid-inducible polypeptides from Eubacterium sp. strain VPI 12708 with molecular weights of 27,000 and approximately 45,000 have previously been shown to be encoded by genes residing on a 2.9-kb EcoRI fragment. We now report the cloning and sequencing of three additional overlapping DNA f ... >> More

  Two bile acid-inducible polypeptides from Eubacterium sp. strain VPI 12708 with molecular weights of 27,000 and approximately 45,000 have previously been shown to be encoded by genes residing on a 2.9-kb EcoRI fragment. We now report the cloning and sequencing of three additional overlapping DNA fragments upstream from this EcoRI fragment. Together, these four fragments contain a large segment of a bile acid-inducible operon which encodes the 27,000- and 45,000-Mr (now shown to be 47,500-Mr) polypeptides and open reading frames potentially coding for four additional polypeptides with molecular weights of 59,500, 58,000, 19,500, and 9,000 to 11,500. A bile acid-inducible polypeptide with an apparent Mr of 23,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to homogeneity, and the N-terminal amino acid sequence that was obtained matched the sequence deduced from the open reading frame coding for the 19,500-Mr polypeptide. A short DNA segment containing the 3' downstream end of the gene coding for the 47,500-Mr polypeptide was not successfully cloned but was directly sequenced from DNA fragments synthesized by polymerase chain reaction. The mRNA initiation site for the bile acid-inducible operon was shown by primer extension to be immediately upstream from the gene encoding the 58,000-Mr polypeptide. A potential promoter region upstream from the mRNA initiation site displayed significant homology with the promoter regions of previously identified bile acid-inducible genes from Eubacterium sp. strain VPI 12708. We hypothesize that this bile acid-inducible operon codes for most of the enzymes involved in the bile acid 7 alpha-dehydroxylation pathway in this bacterium. << Less

  J. Bacteriol. 172:7011-7019(1990) [PubMed] [EuropePMC]

• Expression and characterization of a C24 bile acid 7 alpha-dehydratase from Eubacterium sp. strain VPI 12708 in Escherichia coli.

  Dawson J.A., Mallonee D.H., Bjorkhem I., Hylemon P.B.

  The intestinal bacterium Eubacterium sp. strain VPI 12708 has been shown to have a bile acid 7 alpha/7 beta-dehydroxylation pathway. A large bile acid inducible (bai) operon encoding at least 9 open reading frames has been cloned and sequenced from this bacterium. The baiE gene from this operon ha ... >> More

  The intestinal bacterium Eubacterium sp. strain VPI 12708 has been shown to have a bile acid 7 alpha/7 beta-dehydroxylation pathway. A large bile acid inducible (bai) operon encoding at least 9 open reading frames has been cloned and sequenced from this bacterium. The baiE gene from this operon has been subcloned and expressed in E. coli and found to encode a bile acid 7 alpha-dehydratase (BA7 alpha D). The purified BA7 alpha D was shown to have a calculated subunit mass of 19 kD and a relative native molecular weight of 36,000. The Km and Vmax for 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acid was 0.16 mM and 0.48 nmol/min per mg protein, respectively. Of the substrates tested, the BA7 alpha D used only 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acid and 7 alpha-hydroxy-3-oxo-4-cholenoic acid as substrates. A molecular modeling program (SYBYL) was used to calculate the energy differences between the various intermediates in the 7 alpha-dehydroxylation pathway. A marked energy difference (-9.4 kcal/mol) was observed between 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acid and 12 alpha-hydroxy-3-oxo-4,6-choldienoic acid, possibly accounting for the apparent irreversibility of the bile acid 7 alpha-dehydratase reaction under our experimental conditions. No significant amino acid sequence homologies were found between BA7 alpha D and other proteins in the data base; however, BA7 alpha D does contain a lipocalin signature sequence, possibly indicating a bile acid binding domain. The bile acid 7 alpha-dehydratase appears to be a unique enzyme in the bacterial bile acid 7 alpha-dehydroxylation pathway. << Less

  J. Lipid Res. 37:1258-1267(1996) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structure and functional characterization of a bile acid 7alpha dehydratase BaiE in secondary bile acid synthesis.

  Bhowmik S., Chiu H.P., Jones D.H., Chiu H.J., Miller M.D., Xu Q., Farr C.L., Ridlon J.M., Wells J.E., Elsliger M.A., Wilson I.A., Hylemon P.B., Lesley S.A.

  Conversion of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is performed by a few species of intestinal bacteria in the genus Clostridium through a multistep biochemical pathway that removes a ... >> More

  Conversion of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is performed by a few species of intestinal bacteria in the genus Clostridium through a multistep biochemical pathway that removes a 7α-hydroxyl group. The rate-determining enzyme in this pathway is bile acid 7α-dehydratase (baiE). In this study, crystal structures of apo-BaiE and its putative product-bound [3-oxo-Δ(4,6) -lithocholyl-Coenzyme A (CoA)] complex are reported. BaiE is a trimer with a twisted α + β barrel fold with similarity to the Nuclear Transport Factor 2 (NTF2) superfamily. Tyr30, Asp35, and His83 form a catalytic triad that is conserved across this family. Site-directed mutagenesis of BaiE from Clostridium scindens VPI 12708 confirm that these residues are essential for catalysis and also the importance of other conserved residues, Tyr54 and Arg146, which are involved in substrate binding and affect catalytic turnover. Steady-state kinetic studies reveal that the BaiE homologs are able to turn over 3-oxo-Δ(4) -bile acid and CoA-conjugated 3-oxo-Δ(4) -bile acid substrates with comparable efficiency questioning the role of CoA-conjugation in the bile acid metabolism pathway. << Less

  Proteins 84:316-331(2016) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A metabolic pathway for bile acid dehydroxylation by the gut microbiome.

  Funabashi M., Grove T.L., Wang M., Varma Y., McFadden M.E., Brown L.C., Guo C., Higginbottom S., Almo S.C., Fischbach M.A.

  The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of ... >> More

  The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of Clostridium difficile¹, promote hepatocellular carcinoma² and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. ³). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids⁴; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool. << Less

  Nature 582:566-570(2020) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.