Publications

• Molecular cloning and expression of rat liver bile acid CoA ligase.

  Falany C.N., Xie X., Wheeler J.B., Wang J., Smith M., He D., Barnes S.

  Bile acid CoA ligase (BAL) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5'-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa ... >> More

  Bile acid CoA ligase (BAL) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5'-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa protein with a molecular mass of 75,960 Da, and a 138 nucleotide 3'-nontranslated region followed by a poly(A) tail. Identity of the cDNA was established by: 1) the rBAL-1 open reading frame encoded peptides obtained by chemical sequencing of the purified rBAL protein; 2) expressed rBAL-1 protein comigrated with purified rBAL during SDS-polyacrylamide gel electrophoresis; and 3) rBAL-1 expressed in insect Sf9 cells had enzymatic properties that were comparable to the enzyme isolated from rat liver. Evidence for a relationship between fatty acid and bile acid metabolism is suggested by specific inhibition of rBAL-1 by cis-unsaturated fatty acids and its high homology to a human very long chain fatty acid CoA ligase. In summary, these results indicate that the cDNA for rat liver BAL has been isolated and expression of the rBAL cDNA in insect Sf9 cells results in a catalytically active enzyme capable of utilizing several different bile acids as substrates. << Less

  J. Lipid Res. 43:2062-2071(2002) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Participation of two members of the very long-chain acyl-CoA synthetase family in bile acid synthesis and recycling.

  Mihalik S.J., Steinberg S.J., Pei Z., Park J., Kim do G., Heinzer A.K., Dacremont G., Wanders R.J., Cuebas D.A., Smith K.D., Watkins P.A.

  Bile acids are synthesized de novo in the liver from cholesterol and conjugated to glycine or taurine via a complex series of reactions involving multiple organelles. Bile acids secreted into the small intestine are efficiently reabsorbed and reutilized. Activation by thioesterification to CoA is ... >> More

  Bile acids are synthesized de novo in the liver from cholesterol and conjugated to glycine or taurine via a complex series of reactions involving multiple organelles. Bile acids secreted into the small intestine are efficiently reabsorbed and reutilized. Activation by thioesterification to CoA is required at two points in bile acid metabolism. First, 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid, the 27-carbon precursor of cholic acid, must be activated to its CoA derivative before side chain cleavage via peroxisomal beta-oxidation. Second, reutilization of cholate and other C24 bile acids requires reactivation prior to re-conjugation. We reported previously that homolog 2 of very long-chain acyl-CoA synthetase (VLCS) can activate cholate (Steinberg, S. J., Mihalik, S. J., Kim, D. G., Cuebas, D. A., and Watkins, P. A. (2000) J. Biol. Chem. 275, 15605-15608). We now show that this enzyme also activates chenodeoxycholate, the secondary bile acids deoxycholate and lithocholate, and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid. In contrast, VLCS activated 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate, but did not utilize any of the C24 bile acids as substrates. We hypothesize that the primary function of homolog 2 is in the reactivation and recycling of C24 bile acids, whereas VLCS participates in the de novo synthesis pathway. Results of in situ hybridization, topographic orientation, and inhibition studies are consistent with the proposed roles of these enzymes in bile acid metabolism. << Less

  J. Biol. Chem. 277:24771-24779(2002) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Actinobacterial acyl coenzyme A synthetases involved in steroid side-chain catabolism.

  Casabon I., Swain K., Crowe A.M., Eltis L.D., Mohn W.W.

  Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are p ... >> More

  Bacterial steroid catabolism is an important component of the global carbon cycle and has applications in drug synthesis. Pathways for this catabolism involve multiple acyl coenzyme A (CoA) synthetases, which activate alkanoate substituents for β-oxidation. The functions of these synthetases are poorly understood. We enzymatically characterized four distinct acyl-CoA synthetases from the cholate catabolic pathway of Rhodococcus jostii RHA1 and the cholesterol catabolic pathway of Mycobacterium tuberculosis. Phylogenetic analysis of 70 acyl-CoA synthetases predicted to be involved in steroid metabolism revealed that the characterized synthetases each represent an orthologous class with a distinct function in steroid side-chain degradation. The synthetases were specific for the length of alkanoate substituent. FadD19 from M. tuberculosis H37Rv (FadD19Mtb) transformed 3-oxo-4-cholesten-26-oate (kcat/Km = 0.33 × 10(5) ± 0.03 × 10(5) M(-1) s(-1)) and represents orthologs that activate the C8 side chain of cholesterol. Both CasGRHA1 and FadD17Mtb are steroid-24-oyl-CoA synthetases. CasG and its orthologs activate the C5 side chain of cholate, while FadD17 and its orthologs appear to activate the C5 side chain of one or more cholesterol metabolites. CasIRHA1 is a steroid-22-oyl-CoA synthetase, representing orthologs that activate metabolites with a C3 side chain, which accumulate during cholate catabolism. CasI had similar apparent specificities for substrates with intact or extensively degraded steroid nuclei, exemplified by 3-oxo-23,24-bisnorchol-4-en-22-oate and 1β(2'-propanoate)-3aα-H-4α(3″-propanoate)-7aβ-methylhexahydro-5-indanone (kcat/Km = 2.4 × 10(5) ± 0.1 × 10(5) M(-1) s(-1) and 3.2 × 10(5) ± 0.3 × 10(5) M(-1) s(-1), respectively). Acyl-CoA synthetase classes involved in cholate catabolism were found in both Actinobacteria and Proteobacteria. Overall, this study provides insight into the physiological roles of acyl-CoA synthetases in steroid catabolism and a phylogenetic classification enabling prediction of specific functions of related enzymes. << Less

  J. Bacteriol. 196:579-587(2014) [PubMed] [EuropePMC]

  This publication is cited by 5 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.

• The bile acid-inducible baiB gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A ligase.

  Mallonee D.H., Adams J.L., Hylemon P.B.

  The baiB gene from Eubacterium sp. strain VPI 12708 was previously cloned, sequenced, and shown to be part of a large bile acid-inducible operon encoding polypeptides believed to be involved in bile acid 7 alpha-dehydroxylation. In the present study, the baiB gene was subcloned and expressed in Es ... >> More

  The baiB gene from Eubacterium sp. strain VPI 12708 was previously cloned, sequenced, and shown to be part of a large bile acid-inducible operon encoding polypeptides believed to be involved in bile acid 7 alpha-dehydroxylation. In the present study, the baiB gene was subcloned and expressed in Escherichia coli and shown to encode a bile acid-coenzyme A (CoA) ligase. This ligase required a C-24 bile acid with a free carboxyl group, ATP, Mg2+, and CoA for synthesis of the final bile acid-CoA conjugate. Product analysis by reverse-phase high-performance liquid chromatography revealed final reaction products that comigrated with cholyl-CoA and AMP. A putative bile acid-AMP intermediate was detected when CoA was omitted from the reaction mixture. The bile acid-CoA ligase has amino acid sequence similarity to several other polypeptides involved in the ATP-dependent linking of AMP or CoA to cyclic carboxylated compounds. The bile acid-CoA ligation is believed to be the initial step in the bile acid 7 alpha-dehydroxylation pathway in Eubacterium sp. strain VPI 12708. << Less

  J. Bacteriol. 174:2065-2071(1992) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Determination of the mechanism of reaction for bile acid: CoA ligase.

  Kelley M., Vessey D.A.

  The reaction of cholic acid, CoA and ATP to yield cholyl-CoA was investigated by kinetic analysis of the reaction as catalysed by guinea pig liver microsomes. The enzyme has an absolute requirement for divalent cation for activity so all kinetic analyses were carried out in excess Mn2+. A trisubst ... >> More

  The reaction of cholic acid, CoA and ATP to yield cholyl-CoA was investigated by kinetic analysis of the reaction as catalysed by guinea pig liver microsomes. The enzyme has an absolute requirement for divalent cation for activity so all kinetic analyses were carried out in excess Mn2+. A trisubstrate kinetic analysis was conducted by varying, one at a time ATP cholate and CoA. Both ATP and cholate gave parallel double reciprocal plots versus CoA, which indicates a ping-pong mechanism with either pyrophosphate or AMP leaving prior to the binding of CoA. Addition of pyrophosphate to the assays changed the parallel plots to intersecting ones; addition of AMP did not. This indicates that pyrophosphate is the first product. The end-product, AMP, was a competitive inhibitor versus ATP, as was cholyl-CoA at saturating concentrations of cholate. Both AMP and cholyl-CoA were uncompetitive inhibitors versus CoA. Based on this information, it was concluded that the reaction follows a bi uni uni bi ping-pong mechanism with ATP binding first, and with the release of the final products, AMP and cholyl-CoA, being random. CoA showed substrate inhibition at high but non-saturating concentrations and this inhibition was competitive versus ATP, which is consistent with the predicted ping-pong mechanism. The ability of cholyl-CoA, but not cholate or CoA, to bind with high affinity to the free enzyme was suggestive of a high affinity of the enzyme for the thioester link. << Less

  Biochem J 304:945-949(1994) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.