Molecular cloning of bile acid 7-dehydroxylase from Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is a human intestinal bacterium which contains an inducible bile acid 7-dehydroxylase. Two-dimensional polyacrylamide gel electrophoresis showed that at least four new polypeptides were synthesized after exposure of growing cells to sodium cholate. One of these, of molecular weight 27,000 (PP-27), was implicated in 7-dehydroxylase catalysis. PP-27 was purified to greater than 95% homogeneity by DEAE-cellulose chromatography, high-pressure liquid chromatographic gel filtration, high-pressure liquid chromatography-DEAE chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 33 amino acid residues of the N terminus of PP-27 were determined with a gas-phase sequencer, and a corresponding mixed oligonucleotide (17-mer) was synthesized. Southern blot analysis of EcoRI total digests of chromosomal DNA showed a 2.2-kilobase fragment which hybridized to the 32P-labeled 17-mer. This fragment was enriched for by size fractionation of an EcoRI total digest of genomic DNA, ligated into the bacterial plasmid pUC8, and used to transform Escherichia coli HB101. Transformants containing the putative 7-dehydroxylase gene were detected with the 32P-labeled 17-mer by colony hybridization techniques. The insert was 2.2 kilobases in length and contained the first 290 bases of the PP-27 gene. Preliminary nucleic acid sequence data correlate with the amino acid sequence. The entire gene was cloned on a 1,150-base-pair TaqI fragment. Western blot analysis of E. coli strains containing these plasmids indicated that PP-27 is expressed in E. coli but is not regulated by bile acids under the conditions used.     

Evidence for a multigene family involved in bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is a human intestinal isolate which has an inducible bile acid 7-dehydroxylation activity. At least two cholic acid-induced polypeptides, with molecular masses of 27,000 and 45,000 daltons, respectively, coelute with bile acid 7-dehydroxylation activity. The 45,000-dalton polypeptide appears to be encoded by a cholic acid-induced mRNA species of greater than 6 kilobases, which suggests that the gene coding for this polypeptide is part of a larger operon. A gene has been cloned which flanks the gene encoding the 45,000-dalton polypeptide, in the upstream (5') direction. This gene appears to encode a second 27,000-dalton polypeptide. The gene bears striking homology at both the nucleotide (80%) and deduced amino acid sequence (89%) levels with the gene which encodes the 27,000-dalton polypeptide that has been shown previously to be involved in the bile acid 7-dehydroxylation reaction sequence. The implications of this homology and the possible function(s) of the two homologous genes in bile acid 7-dehydroxylation are discussed. Evidence is presented which suggests that the two homologous genes involved in bile acid 7-dehydroxylation may be part of a larger multigene family in Eubacterium sp. strain VPI 12708.     

Nucleotide sequence and regulation of a gene involved in bile acid 7-dehydroxylation by Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium that has inducible bile acid 7-dehydroxylation activity. At least four new polypeptides were synthesized after addition of primary bile acids to the growth medium. One of these, of molecular weight 27,000 (P-27), was shown to be involved in the 7-dehydroxylation reaction sequence. The gene coding for P-27 was cloned, and the entire DNA sequence for the protein-coding region was determined. In addition, sequence information was obtained for 294 bases upstream from the translational start codon and 329 bases downstream from the stop codon. Induction studies with a synthetic oligonucleotide probe (16-mer) revealed the presence of a cholic acid-inducible mRNA species approximately 900 bases long. A 5' terminus of this mRNA was detected by primer extension analysis, and the location of the 3' terminus of the mRNA was estimated by using S1 nuclease mapping. The 3' terminus of the mRNA contained a large element with dyad symmetry of unknown function. The open reading frame contained 249 codons, and the corresponding polypeptide had a calculated molecular weight of 26,745. The amino acid sequence of P-27 showed significant homology to several previously described alcohol-polyol dehydrogenases ("nonzinc" dehydrogenases), especially in the region believed to contain a pyridine nucleotide-binding domain. The implications of this homology and the possible function of P-27 in bile acid 7-dehydroxylation are discussed.     

HPLC purification and preparation of antibodies to cholic acid-inducible polypeptides from Eubacterium sp. V.P.I. 12708

The role of bile acid-inducible polypeptides in 7-dehydroxylation was investigated in Eubacterium sp. V.P.I. 12708. Cholic acid-inducible bile acid 7 alpha-, 7 beta-dehydroxylase, and delta 6 reductase activities co-eluted from a gel filtration high performance liquid chromatography (HPLC) column. Antibody (Ab) was prepared to these enzymatically active fractions, immunoadsorbed with uninduced cell extract coupled to Sepharose 4B, and used for immunoprecipitation of [35S]-methionine-labeled polypeptides. Ab immunoprecipitated polypeptides with molecular weights of 45,000, 27,000, and 23,500 from induced but not uninduced cell extracts. Immunoinhibition experiments showed that this Ab preparation inhibited (60%) bile acid 7 alpha-dehydroxylase activity in cell extracts. The 45,000 mol wt polypeptide was purified by (NH4)2SO4 fractionation, HPLC gel filtration, and HPLC-DEAE chromatography. Ab prepared to the 45,000 mol wt polypeptide immunoprecipitated only that polypeptide. This Ab, however, did not inhibit bile acid 7 alpha-dehydroxylase activity. Ab specific for the 27,000 mol wt polypeptide was prepared by partial purification and immunoadsorption with uninduced cell extracts. Immunochemical staining, following SDS-PAGE of crude cell extracts, shows a single immunoreactive protein band at 27,000 daltons. This Ab immunoprecipitated the 27,000 mol wt polypeptide as well as small amounts of the 45,000 and 23,000 mol wt polypeptides. Immunoinhibition studies showed that this Ab preparation inhibited (25%) 7 alpha-dehydroxylase activity. These data suggest that the 27,000 mol wt polypeptide is involved in enzyme catalysis. This does not, however, eliminate some role for the 45,000 and 23,500 mol wt polypeptides in bile acid metabolism in this organism.     

Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium.     

Molecular cloning of a gene encoding a 45,000-dalton polypeptide associated with bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an intestinal anaerobic bacterium which possesses an inducible bile acid 7-dehydroxylation activity. Two cholic acid-induced polypeptides with apparent molecular weights of 27,000 and 45,000, respectively, coeluted with bile acid 7-dehydroxylation activity upon anaerobic high-performance gel filtration chromatography of crude cellular protein extracts. The 45,000-dalton polypeptide was purified to greater than 95% homogeneity by high-performance liquid chromatography gel filtration and high-performance liquid-DEAE chromatography. The first 28 amino acid residues of the N terminus of this polypeptide were determined by gas-phase sequencing, and a corresponding mixed oligonucleotide (20-mer) was synthesized. Southern blot analysis of EcoRI total digests of chromosomal DNA showed a 2.6-kilobase fragment which hybridized to the 32P-labeled 20-mer. This fragment was enriched for by size fractionation of an EcoRI total digest of genomic DNA and ligated into bacteriophage lambda gt11. Recombinant phage containing the putative gene encoding the 45,000-dalton polypeptide were detected with the 32P-labeled 20-mer by plaque hybridization techniques. The insert was 2.6 kilobases in length and may contain the entire coding sequence for the 45,000-dalton polypeptide. The 2.6-kilobase insert was subcloned into pUC8 and transformed into Escherichia coli DH5 alpha. However, the 45,000-dalton polypeptide was not detected in cell extracts of this organism when specific antibody was used. Preliminary nucleic acid sequence data correlated exactly with the amino acid sequence. A cholic acid-induced mRNA species of greater than 6 kilobases in size was identified by Northern (RNA) blot analysis of total RNA, suggesting that the gene coding for this polypeptide is part of a larger operon.     

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

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.     

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

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.     

Characterization of the baiH gene encoding a bile acid-inducible NADH:flavin oxidoreductase from Eubacterium sp. strain VPI 12708.

A cholate-inducible, NADH-dependent flavin oxidoreductase from the intestinal bacterium Eubacterium sp. strain VPI 12708 was purified 372-fold to apparent electrophoretic homogeneity. The subunit and native molecular weights were estimated to be 72,000 and 210,000, respectively, suggesting a homotrimeric organization. Three peaks of NADH:flavin oxidoreductase activity (forms I, II, and III) eluted from a DEAE-high-performance liquid chromatography column. Absorption spectra revealed that purified form III, but not form I, contained bound flavin, which dissociated during purification to generate form I. Enzyme activity was inhibited by sulfhydryl-reactive compounds, acriflavine, o-phenanthroline, and EDTA. Activity assays and Western blot (immunoblot) analysis confirmed that expression of the enzyme was cholate inducible. The first 25 N-terminal amino acid residues of purified NADH:flavin oxidoreductase were determined, and a corresponding oligonucleotide probe was synthesized for use in cloning of the associated gene, baiH. Restriction mapping, sequence data, and RNA blot analysis suggested that the baiH gene was located on a previously described, cholate-inducible operon > or = 10 kb long. The baiH gene encoded a 72,006-Da polypeptide containing 661 amino acids. The deduced amino acid sequence of the baiH gene was homologous to that of NADH oxidase from Thermoanaerobium brockii, trimethylamine dehydrogenase from methylotrophic bacterium W3A1, Old Yellow Enzyme from Saccharomyces carlsbergensis, and the product of the baiC gene of Eubacterium sp. strain VPI 12708, located upstream from the baiH gene in the cholate-inducible operon. Alignment of these five sequences revealed potential ligands for an iron-sulfur cluster, a putative flavin adenine dinucleotide-binding domain, and two other well-conserved domains of unknown function.     

Synthesis of bile acid analogs: 7-alkylated chenodeoxycholic acids

This paper describes a method for the preparation of 7-alkylated chenodeoxycholic acids from 3 alpha-hydroxy-7-oxo-5 beta-cholanoic acid. The synthetic procedure is based upon a Grignard reaction between the keto bile acid and an alkyl magnesium halide. Under the conditions employed, the introduction of alkyl groups is highly stereoselective. Only 7 beta-alkylated epimers are obtained. The overall yield is several-fold higher than that obtained by the previous method, which involved the preparation of an oxazoline intermediate.     

Biotransformation of linoleic acid and bile acids by Eubacterium lentum.

Eubacterium lentum is a gram-positive, nonsporeforming, nonmotile, asaccharolytic anaerobe. In the present investigations, 3 E. lentum strains (group E) isolated from rat feces were compared with 30 E. lentum strains (groups A, B, C, and D) previously studied by Macdonald et al. (I. A. Macdonald, J. F. Jellet, D. E. Mahony, and L. V. Holdeman, Appl. Environ. Microbiol. 37:992-1000, 1979). All strains alkalized (pH 8 to 8.5) arginine-containing (2 to 15 mg/ml) culture media, and growth of the majority of the strains was stimulated by arginine. All strains converted linoleic acid into transvaccenic acid by shifting the 12,13-cis double bond of linoleic acid into an 11,12-trans(?) double bond followed by biohydrogenation of the 9,10-cis double bond. Hence, biohydrogenation of linoleic acid is a new general characteristic of E. lentum. The 33 strains were also studied for bile acid deconjugase and hydroxysteroid dehydrogenase (HSDH) activities. The 6 strains in group D were steroid inactive; the 27 strains in groups A, B, C, and E were steroid active. The steroid-active group contained bile acid deconjugase-producing strains (groups C and E, plus strain 116 in group A) and nondeconjugating strains. All nondeconjugating strains of groups A and B developed 7 alpha- and 12 alpha-HSDH activities and contained 3 alpha-HSDH-positive strains and 3 alpha-HSDH-negative strains. Deconjugating strains varied in HSDH activities.     

Bile acid sulfonate and 7-alkylated bile acid analogs: effect on intestinal absorption of taurocholate and cholesterol 7alpha-hydroxylase activity in cultured rat hepatocytes

The effects of sulfonate analogs of cholic (C), chenodeoxycholic (CDC), and ursodeoxycholic acid (UDC) and three 7-alkylated CDCs--7-methyl-, 7-ethyl-, and 7-propyl-CDCs--on taurocholate absorption from rat terminal ileum in situ and on cholesterol 7alpha-hydroxylase activity in primary culture of the rat liver were investigated. The sulfonate analogs of two dihydroxy bile acids CDC and UDC, but not C, significantly decreased the absorption of taurocholate. Taurine conjugates of 7-alkylated CDC slightly decreased the taurocholate absorption, and tauro-7-propyl-CDC significantly suppressed the absorption. Although the sulfonate analogs of C and CDC reduced cholesterol 7alpha-hydroxylase activity by 40% and 60% compared to control, UDC-sulfonate analog did not affect enzymatic activity. These results were consistent with those of the lead compounds, C, CDC, and UDC. The introduction of methyl group at C-7 position of CDC attenuated the reduction in cholesterol 7alpha-hydroxylase activity by CDC. However, elongation of the alkyl group resulted in an inhibitory effect. The present study revealed the following: 1) bile acid sulfonates act on cholesterol and bile acid metabolism in a similar manner as taurine conjugated bile acids; and 2) the biologic properties of CDC could be altered by the introduction of alkyl group at C-7 position.     

Regulation of bile acid synthesis. V. Inhibition of conversion of 7-dehydrocholesterol to cholesterol is associated with down-regulation of cholesterol 7 alpha-hydroxylase activity and inhibition of bile acid synthesis.

In the chronic bile fistula rat, the administration of a bolus dose of mevinolinic acid, an inhibitor of HMG-CoA reductase, was followed by rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and a decrease in bile acid synthesis. These observations suggested that either newly synthesized cholesterol or some other metabolite of mevalonate may be involved in the regulation of bile acid synthesis. In order to distinguish between these two alternatives, we carried out experiments in which cholesterol synthesis was blocked by AY9944, a compound that inhibits the conversion of 7-dehydrocholesterol to cholesterol, a last step in the cholesterol biosynthesis pathway. Rats underwent biliary diversion for 72 h at which time they were given intravenously either a bolus dose of AY9944 (1 mg/kg) or control vehicle. At 0 (pre-treatment control), 0.5, 1.5, and 3 h post bolus, livers were harvested and specific activities of cholesterol 7 alpha-hydroxylase were determined. At 1.5, 3, and 6 h post bolus, AY9944 inhibited bile acid synthesis by 19 +/- 6%, 40 +/- 4%, and 41 +/- 6%, respectively, as compared to pretreatment baseline. Cholesterol 7 alpha-hydroxylase activity determined at 0.5, 1.5, and 3 h was decreased by 44 +/- 6%, 44 +/- 2%, and 36 +/- 2%, respectively, as compared to the control value. In in vitro experiments using microsomes from livers of control bile fistula rats, the addition of AY9944 (up to 100 microM) failed to inhibit cholesterol 7 alpha-hydroxylase activity. The results of this study demonstrate that, in the chronic bile fistula rat, acute inhibition of cholesterol synthesis at either early or late steps leads to a rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and decrease in bile acid synthesis.     

Epidemiological analyses on animal parasitoses: recent activity of the I.M.I.P.P.V

The paper presents a selected review of the epidemiological analyses performed in recent years (1984-1988) in the Institute of Malattie Infettive, Profilassi e Polizia Veterinaria of the University of Bologna on animal parasitoses, with special reference to the evaluation of some risk factors of bovine, swine and canine helminthoses, and to the validity of the coprological test for some parasites of the red fox.     

Biosynthesis of a novel bile acid nucleotide and mechanism of 7 alpha-dehydroxylation by an intestinal Eubacterium species

Eubacterium species V.P.I. 12708 has inducible bile acid 7-dehydroxylase activity that can use either 7 alpha or 7 beta bile acids as substrates. Cell extracts prepared from bacteria grown in the presence of cholic acid catalyzed the rapid conversion of free bile acids into a highly polar bile acid metabolite (HPBA). This conjugation activity co-eluted with bile acid 7-dehydroxylase activity on high performance gel filtration chromatography (GFC). The HPBA was purified by a combination of high performance GFC and reverse-phase high performance liquid chromatography (HPLC). The intact HPBA eluted earlier from reverse-phase HPLC than deoxycholyl-CoA and had a Mr of 1102 by Bio-Gel P-2 (GFC). The HPBA had an absorption peak at 255 nm and was sensitive to treatment with phosphodiesterase I or nucleotide pyrophosphatase. The HPBA has a free phosphate as shown by an increase in elution volume on reverse-phase HPLC following treatment with alkaline phosphatase. Treatment of the purified HPBA with nucleotide pyrophosphate plus alkaline phosphatase yielded adenosine, whereas, treatment with nucleotide pyrophosphatase alone generated 5',3'-ADP. A bile acid metabolite was also generated by nucleotide pyrophosphatase treatment. The bile acid metabolite had different chromatographic properties (HPLC and TLC) than the corresponding free bile acid. Gas liquid chromatography-mass spectrometry showed the bile acid metabolite to be 12 alpha-hydroxy-3-oxo-4-cholenoic acid. We hypothesize that the HPBA is an intermediate in 7-dehydroxylation and consists of this compound linked at the C-24 with an anhydride bond to the beta phosphate (5') of ADP-3'-phosphate. These results suggest a novel mechanism of bile acid 7 alpha/7 beta-dehydroxylation in Eubacterium sp. V.P.I. 12708.     

Maintenance of bile acid synthesis and cholesterol 7 alpha-hydroxylase activity in cultured rat hepatocytes.

Addition of foetal-bovine serum to rat hepatocytes cultured in Williams E medium resulted in improved maintenance of bile-acid-synthetic capacity and cholesterol 7 alpha-hydroxylase activity as compared with cultures supplemented with rat or newborn-bovine serum or cultures in a hormonally defined serum-free medium. Minimally, 5% (v/v) foetal-bovine serum was necessary to maintain these liver-specific functions. Serum factor(s) responsible for these effects were not dialysable or associated with lipoproteins, but were removed by charcoal extraction.     

Effect of bile acid oxazoline derivatives on microorganisms participating in 7 alpha-hydroxyl epimerization of primary bile acids

We tested bile acid oxazoline derivatives of chenodeoxycholic (CDC-OX), 7-ketolithocholic (7-KLC-OX), ursodeoxycholic (UDC-OX), and deoxycholic (DC-OX) as inhibitors of the 7-epimerization of the primary bile acids cholic acid (CA) and CDC in cultures of four species of bacteria and the human fecal flora. The organisms tested elaborate a 7 alpha- and/or 7 beta-hydroxysteroid dehydrogenase (HSDH); they were Escherichia coli (7 alpha-HSDH), Bacteroides fragilis (7 alpha-HSDH), Clostridium absonum (7 alpha- and 7 beta-HSDH) and Eubacterium aerofaciens (7 beta-HSDH). None of the oxazolines affected 7 alpha-OH oxidation of CA or CDC by E. coli or the growth of the organism. All the oxazolines (except UDC-OX) inhibited the growth of B. fragilis and its 7 alpha-HSDH. In contrast, only DC-OX blocked 7 alpha-OH epimerization of CA by C. absonum. Surprisingly, the other three oxazolines enhanced 7 alpha-OH epimerization of CA, but not that of CDC, which was inhibited (CDC-OX greater than 7-KLC-OX much greater than UDC-OX). Enzymic data suggest that CDC-OX in the presence of CA can induce a greater level of both 7 alpha- and 7 beta-HSDH than CA or CDC-OX alone, CDC-OX being more toxic in the presence of CDC. Formation of urso-bile acid from 7-keto substrates by E. aerofaciens is totally blocked by the oxazolines (except UDC-OX). Similarly, suppression of urso-bile acid formation from primary bile acids by the human fecal flora was evident with DC-OX greater than 7-KLC-OX greater than CDC-OX much greater than UDC-OX, the last being ineffective. The inhibitory activity of the oxazolines on the 7-dehydroxylation of primary bile acids by human fecal flora followed the same order.     

Structure-activity relationship of bile acids and bile acid analogs in regard to FXR activation

The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor that plays a major role in bile acid and cholesterol metabolism. To obtain an insight into the structure-activity relationships of FXR ligands, we investigated the functional roles of structural elements in the physiological ligands chenodeoxycholic acid [CDCA; (3alpha,7alpha)], cholic acid [CA; (3alpha,7alpha,12alpha)], deoxycholic acid [DCA; (3alpha,12alpha)], and lithocholic acid (3alpha) in regard to FXR activation in a cell-based FXR response element-driven luciferase assay and an in vitro coactivator association assay. Conversion of the carboxyl group of CDCA or CA to an alcohol did not greatly diminish their ability to activate FXR. In contrast, the 7beta-epimers of the alcohols were inactive, indicating that the bile alcohols retained the ligand properties of the original bile acids and that the 7beta-hydroxyl group diminished their FXR-activating effect. Similarly, hydroxyl epimers of DCA exhibited decreased activity compared with DCA, indicating a negative effect of 3beta- or 12beta-hydroxyl groups. Introduction of an alkyl group at the 7beta- or 3beta-position of CDCA resulted in diminished FXR activation in the following order of alkyl groups: 7-ethyl=7-propyl>3-methyl>7-methyl. These results indicate that bulky substituents, whether hydroxyl groups or alkyl residues, at the beta-position of cholanoids decrease their ability to activate FXR.