The bile acid-inducible baiF gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A hydrolase
1999; Elsevier BV; Volume: 40; Issue: 1 Linguagem: Inglês
10.1016/s0022-2275(20)33335-6
ISSN1539-7262
AutoresHua-Qing Ye, Darrell H. Mallonee, James E. Wells, Ingemar Björkhem, Phillip B. Hylemon,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoThe human intestinal Eubacterium sp. strain VPI 12708 has been shown to have a multistep biochemical pathway for bile acid 7α-dehydroxylation. A bile acid-inducible operon encoding 9 open reading frames has been cloned and sequenced from this organism. Several of the genes in this operon have been shown to catalyze specific reactions in the 7α-dehydroxylation pathway. The baiF gene from this operon was cloned, expressed in Escherichia coli, and found to encode a novel bile acid-coenzyme A (CoA) hydrolase. The subunit molecular mass of the purified bile acid-CoA hydrolase was calculated to be 47,466 daltons and the native enzyme had a relative molecular weight of 72,000. The Km and Vmax for cholyl-coenzyme A (CoA) hydrolysis was approximately 175 μm and 374 μmol/min per mg protein, respectively. The enzyme used cholyl-CoA, 3-dehydrocholyl-CoA, and chenodeoxycholyl-CoA as substrates. No hydrolytic activity was detected using acetyl-CoA, isovaleryl-CoA, palmitoyl-CoA, or phenylacetyl-CoA as substrates. Amino acid sequence database searches showed no significant similarity of bile acid-CoA hydrolase to other thioesterases, but significant amino acid sequence identity was found with Escherichia coli carnitine dehydratase. The characteristic thioesterase active site Gly-X-Ser-X-Gly motif was not found in the amino acid sequence of this enzyme. Bile acid-CoA hydrolase from Eubacterium sp. strain VPI 12708 may represent a new family of thioesterases.—Ye, H-Q., D. H. Mallonee, J. E. Wells, I. Björkem, and P. B. Hylemon. The bile acid-inducible baiF gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A hydrolase. J. Lipid Res. 1999. 40: 17–23. The human intestinal Eubacterium sp. strain VPI 12708 has been shown to have a multistep biochemical pathway for bile acid 7α-dehydroxylation. A bile acid-inducible operon encoding 9 open reading frames has been cloned and sequenced from this organism. Several of the genes in this operon have been shown to catalyze specific reactions in the 7α-dehydroxylation pathway. The baiF gene from this operon was cloned, expressed in Escherichia coli, and found to encode a novel bile acid-coenzyme A (CoA) hydrolase. The subunit molecular mass of the purified bile acid-CoA hydrolase was calculated to be 47,466 daltons and the native enzyme had a relative molecular weight of 72,000. The Km and Vmax for cholyl-coenzyme A (CoA) hydrolysis was approximately 175 μm and 374 μmol/min per mg protein, respectively. The enzyme used cholyl-CoA, 3-dehydrocholyl-CoA, and chenodeoxycholyl-CoA as substrates. No hydrolytic activity was detected using acetyl-CoA, isovaleryl-CoA, palmitoyl-CoA, or phenylacetyl-CoA as substrates. Amino acid sequence database searches showed no significant similarity of bile acid-CoA hydrolase to other thioesterases, but significant amino acid sequence identity was found with Escherichia coli carnitine dehydratase. The characteristic thioesterase active site Gly-X-Ser-X-Gly motif was not found in the amino acid sequence of this enzyme. Bile acid-CoA hydrolase from Eubacterium sp. strain VPI 12708 may represent a new family of thioesterases. —Ye, H-Q., D. H. Mallonee, J. E. Wells, I. Björkem, and P. B. Hylemon. The bile acid-inducible baiF gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A hydrolase. J. Lipid Res. 1999. 40: 17–23. Bile acids are synthesized from cholesterol in the liver. The two primary bile acids synthesized in humans are cholic acid and chenodeoxycholic acid and they are conjugated to either glycine or taurine prior to their active secretion from the liver (1Vlahcevic Z.R. Heuman D.M. Hylemon P.B. Physiology and pathophysiology of enterohepatic circulation of bile acids.in: Zakim D. Boyer T. Hepatology: a Textbook of Liver Diseases. 1. W. B. Saunders, Philadelphia1996: 376-417Google Scholar). They undergo a recycling process between the liver and intestine called enterohepatic circulation. During their enterohepatic circulation several hundred milligrams are lost into the large intestines each day. In the large bowel, a small population of colonic bacteria catalyze 7α-dehydroxylation of cholic acid and chenodeoxycholic acid yielding deoxycholic and lithocholic acid, respectively (2Ferrari A. Pacini N. Canzi E. Brano R. Prevalence of O2-intolerant microorganisms in primary bile acid dehydroxylating mouse intestinal microflora.Curr. Microbiol. 1980; 4: 257-260Google Scholar, 3Stellwag E.J. Hylemon P.B. 7α-Dehydroxylation of cholic acid and chenodeoxycholic acid by Clostridium leptum.J. Lipid Res. 1979; 20: 325-333Google Scholar). Deoxycholic acid is absorbed from the colon and becomes part of the bile acid pool in humans. The amount of deoxycholic acid in the bile acid pool can vary from 0 to over 40% of the total pool (4Berr F. Kulak-Ublick G. Paumgartner G. Münzing W. Hylemon P.B. 7α-Dehydroxylating bacteria enhance deoxycholic acid input and cholesterol saturation of bile in patients with gallstones.Gastroenterology. 1996; 111: 1611-1620Google Scholar). Eubacterium sp. strain VPI 12708 is an anaerobic bacterium originally isolated from a human fecal sample and has been demonstrated to have a bile acid-inducible 7α-dehydroxylation pathway (5Hylemon P.B. Melone P.D. Franklund C.V. Lund E. Björkhem I. Mechanism of intestinal 7α-dehydroxylation ofcholic acid: evidence that allo-deoxycholic acid is an inducible side-product.J. Lipid Res. 1991; 32: 89-96Google Scholar) (Fig. 1). This pathway has been proposed to serve as an ancillary electron acceptor for the fermentative metabolism of this organism. The oxidative part of this pathway is believed to begin by active transport of the primary bile acid into the bacterial cell (6Mallonee D.H. Hylemon P.B. Sequencing and expression of a gene encoding a bile acid transporter from Eubacterium sp. strain VPI 12708.J. Bacteriol. 1996; 178: 7053-7058Google Scholar), followed by synthesis of the coenzyme A (CoA) conjugate (7Mallonee D.H. Adams J.L. Hylemon P.B. The bile acid-inducible baiB gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A ligase.J. Bacteriol. 1992; 174: 2065-2071Google Scholar), oxidation of the 3α-hydroxy group (8Mallonee D.H. Lijewski M.A. Hylemon P.B. Expression in Escherichia coli and characterization of a bile acid-inducible 3α-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.Curr. Microbiol. 1995; 30: 259-263Google Scholar), and insertion of a double bond between C-4 and C-5 (5Hylemon P.B. Melone P.D. Franklund C.V. Lund E. Björkhem I. Mechanism of intestinal 7α-dehydroxylation ofcholic acid: evidence that allo-deoxycholic acid is an inducible side-product.J. Lipid Res. 1991; 32: 89-96Google Scholar). The next step in this pathway involves 7α-dehydration (9Dawson J.A. Mallonee D.H. Björkhem I. Hylemon P.B. Expression and characterization of a C24 bile acid 7α-dehydratase from Eubacterium sp. strain VPI 12708 in Escherichia coli.J. Lipid Res. 1996; 37: 1258-1267Google Scholar), yielding a 3-oxo-Δ4,6-intermediate that is sequentially reduced in three steps to the 7α-dehydroxylated bile acid and released from the cell (Fig. 1). It is not known when the coenzyme A is cleaved from the bile acid. However, the 3α-hydroxysteroid dehydrogenase, an enzyme in the oxidative arm of the pathway, has been shown to strongly prefer bile acid-CoA conjugates as substrates (8Mallonee D.H. Lijewski M.A. Hylemon P.B. Expression in Escherichia coli and characterization of a bile acid-inducible 3α-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.Curr. Microbiol. 1995; 30: 259-263Google Scholar). A large (~12 kb) bile acid-inducible operon has been cloned and sequenced from Eubacterium sp. strain VPI 12708 (10Mallonee D.H. White W.B. Hylemon P.B. Cloning and sequencing of a bile acid-inducible operon from Eubacterium sp. strain VPI 12708.J. Bacteriol. 1990; 172: 7011-7019Google Scholar). This operon has been shown to encode nine open reading frames (baiA to baiI). The function of several of the gene products from this operon in the bile acid 7α-dehydroxylation pathway has been elucidated (Fig. 2).The baiB gene has been shown to encode a bile acid-CoA ligase (7Mallonee D.H. Adams J.L. Hylemon P.B. The bile acid-inducible baiB gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A ligase.J. Bacteriol. 1992; 174: 2065-2071Google Scholar), but how CoA is removed from the bile acid has not been elucidated. We initially hypothesized that the baiF gene may encode a bile acid-CoA hydrolase based on the observations with Escherichia coli extracts. When cell extracts containing the baiF gene product were added to Eubacterium sp. strain VPI 12708 extracts, there was a marked decrease in [14C]cholic acid water-soluble (CoA-conjugated) counts. In this communication, we report the discovery and characterization of a novel bile acid-CoA hydrolase encoded by the baiF gene in the bai operon. Radiolabeled [24-14C]cholic acid and [24-14C]chenodeoxycholic acid were purchased from DuPont NEN (Boston, MA). [24-14C]cholyl-CoA was prepared enzymatically using bile acid-CoA ligase expressed in E. coli essentially as described previously (7Mallonee D.H. Adams J.L. Hylemon P.B. The bile acid-inducible baiB gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A ligase.J. Bacteriol. 1992; 174: 2065-2071Google Scholar). [24-14C]-3-dehydrocholyl-CoA was synthesized enzymatically from [24-14C]cholyl-CoA using 3α-hydroxysteroid dehydrogenase obtained from Sigma Chemical Co. (St. Louis, MO). Acetyl-CoA, isovaleryl-CoA, palmitoyl-CoA, phenylacetyl-CoA were also purchased from Sigma Chemical Co. Cholic acid and chenodeoxycholic acid were obtained from Calbiochem (San Diego, CA). The cholyl-CoA ester was synthesized by the mixed anhydride procedure essentially as described by Shah and Staple (11Shah P.P. Staple E. Synthesis of coenzyme A esters of some bile acids.Steroids. 1968; 12: 571-576Google Scholar). Coupling of 75 μmol cholic acid with 63 μmol CoASH gave 15 μmol product that showed the expected UV-absorption at 232 nm. The purified material was found to contain less than 2% free cholic acid and was stable for several weeks when stored at 4°C in aqueous sodium acetate solution 0.01 m, pH 6.0. E. coli DH5α was obtained from Gibco BRL (Gaithersburg, MD) and used as the host strain for expression of recombinant plasmids. Most of the baiF gene from Eubacterium sp. strain VPI 12708 is contained in a 2.9 kb EcoRI fragment that was initially cloned in E. coli by using a λgt11 vector, and then subcloned into a pUC8 vector (12White W.B. Coleman J.P. Hylemon P.B. Molecular cloning of a gene encoding a 45,000-dalton polypeptide associated with bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.J. Bacteriol. 1988; 170: 611-616Google Scholar). The sequence at the 3′ end of the baiF gene was obtained through direct sequencing of DNA obtained by inverse PCR (10Mallonee D.H. White W.B. Hylemon P.B. Cloning and sequencing of a bile acid-inducible operon from Eubacterium sp. strain VPI 12708.J. Bacteriol. 1990; 172: 7011-7019Google Scholar). A 6 kb fragment, which contained the entire baiF gene, was obtained by BamH1 digestion of chromosomal DNA from Eubacterium sp. strain VPI 12708. This fragment was used as the template for PCR amplification of the baiF gene using the synthetic oligonucleotide primers 5′-CCAGTCGACTTACGGC TACTACGGTCAGCGTAGA-3′ and 5′-CCAGGATCCGGTGCT CATACTCTTACTCCTCTT-3′, which added SalI and BamHI sites, respectively (in bold). The DNA generated from this reaction was run on an agarose gel and the fragment corresponding to the amplified baiF gene was cut out and purified with a GeneClean kit (Bio 101 Inc., La Jolla, Calif). The purified DNA was digested with SalI and BamHI, ligated to pUC19 vector DNA, and transformed into E. coli DH5α. Isolated plasmids with the correct baiF insert were designated pUC19-47K. The baiF insert in pUC19-47K was completely sequenced to verify polymerase fidelity. Expression of the baiF gene in E. coli was confirmed by a Western blot using rabbit polyclonal antibody which recognizes the baiF gene product essentially as described previously (12White W.B. Coleman J.P. Hylemon P.B. Molecular cloning of a gene encoding a 45,000-dalton polypeptide associated with bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.J. Bacteriol. 1988; 170: 611-616Google Scholar). Bile acid-CoA hydrolase activity was assayed by measuring the rate of conversion of [24-14C]cholyl-CoA to free [24-14C]cholic acid and CoA. The standard reaction mixture contained in final concentrations: 20 mm sodium phosphate buffer (pH 7.0); 5,000 dpm [24-14C]cholyl-CoA, 100 μm unlabeled cholyl-CoA, and enzyme preparation in a total volume of 100 μl. The reaction was initiated by the addition of enzyme and carried out at 37°C. The reaction was stopped by the addition of 10 μl of 1 m HC1. Unconjugated cholic acid was extracted with ethyl-acetate phase and radioactivity in both the organic and aqueous phases was quantitated by liquid scintillation spectrometry. The reaction rate was linear for 2 min and over a protein concentration range of 0.01 to 0.12 μg/reaction mixture, using purified enzyme. A control reaction without enzyme was run and the background counts were subtracted. Substrate saturation kinetics were performed in two independent determinations and an average Km and Vmax were determined using a Hanes plot (13Hanes C.S. CLXVII. Studies on plant amylases. I. The effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley.Biochem. J. 1932; 26: 1406-1421Google Scholar). A reversed phase C18 column (4.6 mm by 25 cm) (10-μm silica gel) was used for analysis of bile acid-CoA hydrolase reaction products. Reaction mixtures (20 μl) were loaded onto the column and eluted with 5–40% isopropanol in 20 mm ammonium bicarbonate buffer. Gradients were run over a 40-min time period at a flow rate of 0.8 ml/min. Free CoA and cholyl-CoA conjugates were monitored at 260 nm. Analysis of the bile acid component of cholyl-CoA was carried out on silica gel 1B thin-layer chromatography (TLC) plates (J.T. Baker, Inc., Phillipsburg, NJ). The cholyl-CoA hydrolase reaction was performed with standard reaction mixtures for 10 min at 37°C before being stopped with 1 m HC1. The reaction mixture was extracted with ethyl-acetate, spotted on TLC plates, and chromatographed with solvent system S1 (14Eneroth P. Thin-layer chromatography of bile acids.J. Lipid Res. 1963; 4: 11-16Google Scholar). The plates were dried and exposed to Kodak X-Omat AR film (Eastman Kodak Co., Rochester, NY) for 24 h at room temperature before development. Known bile acid standards were run as controls. Escherichia coli DH5α containing the pUC19-47K plasmid was used to inoculate two liters of Luria Broth (LB) containing ampicillin. This strain was grown aerobically at 37°C to a reading of 80 Klett units (red filter) before induction with 0.5 mm IPTG. After 2 h of additional growth, the induced cells were harvested by centrifugation, resuspended in 50 mm sodium phosphate buffer (pH 7.0) containing 20 mm 2-mercaptoethanol (2 ME) and 100 μg DNase, and broken by passing twice through a French Pressure Cell at 20,000 lb/in2. The cell lysate was centrifuged at 105,000 g for 2 h at 4°C, and the supernatant fluid was collected and concentrated using a Centriprep-10 concentrator (Amicon Corp., Danvers, MA). The concentrated supernatant was diluted in 50% glycerol and stored at −20°C for further use. The soluble cell extract (360 mg) was applied to a Waters AP-2 DEAE high performance liquid chromatography (HPLC) column (Millipore Corp., Burlington, MA) equilibrated with 20 mm sodium phosphate buffer (pH 6.5) containing 20 mm 2 ME. Protein was eluted with a 0 to 500 mm NaCl gradient at a flow rate of 3.5 ml/min. Fractions containing high bile acid-CoA hydrolase activity were pooled, concentrated using a Centriprep-10 concentrator, and stored overnight in 10% glycerol at −20°C. These fractions were then applied to a Mono-Q ion-exchange column (Pharmacia LKB Biotechnology, Piscataway, NJ) equilibrated with 50 mm sodium phosphate buffer (pH 7.0) containing 20 mm 2-ME and 5% glycerol. Bile acid-CoA hydrolase was eluted with a 0 to 500 mm sodium chloride gradient at a flow rate of 1 ml/min using the equilibration buffer. Fractions containing activity were concentrated with a Centricon-10 microconcentrator, brought to 50% glycerol, and stored at −20°C. Amino acid sequence analysis and data base searching was performed with the Wisconsin Package (Genetics Computer Group, Madison, WI). Transformation of the pUC19-47K plasmid into E. coli resulted in excellent expression and accumulation of the baiF gene product (Fig. 3, compare lanes 1 and 2). Cell extracts of E. coli DH5α containing the pUC19-47K plasmid yielded ~330 mg of soluble protein, with baiF protein representing ~15% of the total protein. The expression and identity of the baiF gene product was confirmed by Western blotting using antibody to the 45 kilodalton protein (Fig. 4).Fig. 4Western immunoblot of baiF protein. Two μg of total soluble protein was used per lane for Eubacterium sp. strain VPI 12708 (1), E. coli DH5α pUC19-47K (2), and E. coli DH5α control (3).View Large Image Figure ViewerDownload (PPT) Eubacterium sp. strain VPI 12708 bile acid-CoA hydrolase was purified from E. coli (pUC19-47K) using HPLC. A 13-fold purification with a 38% yield was obtained from a typical two-step purification protocol (Table 1). Samples of each purification step were analyzed by SDS-PAGE (Fig. 3). The final enzyme preparation was greater than 90% pure (Fig. 3, lane 4). The purified enzyme had a subunit molecular mass of ~47,500 daltons as judged by SDS-PAGE. The native relative molecular weight was 72,000 using gel filtration chromatography. The bile acid-CoA hydrolase activity was stable for several months whenstored at −20°C in 50% glycerol. E. coli DH5α (without plasmid) had some endogenous (~7.5% of total activity as compared with pUC19-47K plasmid) bile acid-CoA hydrolase (thioesterase) activity. The endogenous activity was fractionated on DEAE chromatography using identical conditions as described for the purification of bile acid-CoA hydrolase activity encoded by the baiF gene product. The endogenous activity eluted at a higher salt concentration on DEAE chromatography than the bile acid CoA hydrolase activity encoded by the baiF gene (data not shown).TABLE 1Purification of bile acid-CoA hydrolase from E. coliPurification StepTotal ProteinActivityaOne unit of activity is defined as the amount of enzyme required to hydrolyze 1 μmol cholyl-CoA per min under standard assay conditions (see Materials and Methods).Sp. Act.YieldPurificationmgUU/mg%-foldSoluble cell extract36017324.81100DEAE32112635657.3Mono-Q11659613812.68a One unit of activity is defined as the amount of enzyme required to hydrolyze 1 μmol cholyl-CoA per min under standard assay conditions (see Materials and Methods). Open table in a new tab Bile acid-CoA hydrolase activity was measured by determining the rate of formation of ethyl-acetate-soluble (free cholic acid) radioactivity extracted from reaction mixtures containing [24-14C]cholyl-CoA. Using standard reaction mixtures, the catalysis rate was linear for 2 min and over a protein concentration of 0.01 to 0.12 μg protein. Enzymatic activity was optimal between pH 6.0 and 7.0. No activity was detected below pH 4.0 or above pH 9.0. Km and Vvax values were obtained using initial velocity conditions. The approximate Km and Vmax values for cholyl-CoA hydrolysis were 175 μm and 374 μmol/min per mg protein, respectively. Kinetic constants were determined using a Hanes Plot (13Hanes C.S. CLXVII. Studies on plant amylases. I. The effect of starch concentration upon the velocity of hydrolysis by the amylase of germinated barley.Biochem. J. 1932; 26: 1406-1421Google Scholar). Products from the bile acid-CoA hydrolase reaction mixture were analyzed by reversed-phase HPLC and TLC. The elution times for CoA (HSCoA) and cholyl-CoA were determined by monitoring eluant at 260 nm. When purified bile acid-CoA hydrolase was added to standard reaction mixtures containing cholyl-CoA, a rapid accumulation of a product co-migrating with HSCoA was observed (Fig. 5, time 2 and 10 min). When the ethyl-acetate-soluble fractions from these reaction mixtures were analyzed by TLC, a single product co-migrating with cholic acid was observed (data not shown). Using either reversed-phase HPLC or TLC to identify enzymatic products, cholyl-CoA, 3-dehydrocholyl-CoA, and chenodeoxycholyl-CoA were shown to be used as substrates by bile acid CoA hydrolase. However, no hydrolytic activity was detected when acetyl-CoA, isovaleryl-CoA, palmitoyl-CoA, or phenylacetyl-CoA were used as substrates. FASTA and BLAST searches were performed for amino acid sequence homology to bile acid CoA hydrolase. Sequences identified by these homology searches were then aligned with the GAP program. The best overall homology (38.2% identity and 60.1% similarity) was found with carnitine dehydratase from E. coli (15Eichler K. Schunck W-H. Kleber H-P. Mandrand-Berthelot M-A. Cloning, sequencing, and expression of the Escherichia coli gene encoding carnitine dehydratase.J. Bacteriol. 1994; 176: 2970-2975Google Scholar) and Fig. 6. However, the baiF gene product had no bile acid 7α-dehydratase activity using 7α,12α-dihydroxy-3-oxo-4-cholenoic acid as substrate (data not shown). No sequence homology was found with any reported 3-ketoacyl-CoA thiolases, acyl-CoAhydrolases, or thiotransferases. In addition, consensus active site amino acid sequences for serine esterases (16Hardie D.G. Dewart K.B. Aitben A. McCarthy A.D. Amino acid sequence around the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase.Biochim. Biophys. Acta. 1985; 828: 380-382Google Scholar, 17Randhawa Z.I. Naggert J. Blacher R.W. Smith S. Amino acid sequencing of the serine active-site region of the medium-chain S-acyl fatty acid synthetase thioester hydrolase from rat mammary gland.Eur. J. Biochem. 1987; 162: 577-581Google Scholar, 18Tai M-H. Chirala S.S. Wakil S.J. Roles of Ser101, Asp236, and His237 in catalysis of thioesterase II and the C-terminal region of the enzyme in its interaction with fatty acid synthase.Proc. Natl. Acad. Sci. USA. 1993; 90: 1852Google Scholar, 19Witkowski A. Naggert J. Witkowska H.E. Randhawa Z.I. Smith S. Utilization of an active serine 101-cysteine mutant to demonstrate the proximity of the catalytic serine 101 and histidine 237 residues in thioesterase II.J. Biol. Chem. 1992; 267: 18488-18492Google Scholar), CoA transferases/acyl-CoA hydrolases (20Mack M. Buckel W. Conversion of glutaconate CoA-transferase from Acidaminococcus fermentans into an acyl-CoA hydrolase by site-directed mutagenesis.FEBS Lett. 1997; 405: 209-212Google Scholar), and 3-ketoacyl-CoA thiolases (21Bodnar A.G. Rachubinski R.A. Cloning and sequence determination of cDNA encoding a second rat liver peroxisomal 3-keotacyl-CoA thiolase.Gene. 1990; 91: 193-199Google Scholar) could not be found in bile acid coenzyme A hydrolase. Acyl-CoA hydrolase (thioesterase) activities have been reported in various mammalian tissues and in microorganisms. These enzymes are involved in a variety of biochemical processes including: regulation of intracellular fatty acid and acyl-CoA concentrations (22Sanjanwala M. Sun G.Y. MacQuarrie R.A. Purification of long-chain acyl-CoA hydrolase from bovine heart microsomes and regulation of activity by lysophopholipids.Arch Biochem. Biophys. 1987; 258: 299-306Google Scholar); hydrolysis of fatty acids from acyl-carrier protein (23Lin C.Y. Smith S. Properties of the thioesterase component obtained by limited trypsinization of the fatty acid synthetase multienzyme complex.J. Biol. Chem. 1978; 253: 1954-1962Google Scholar); dehalogenation of chlorinated aromatic compounds in bacteria (24Dunaway-Marino D. Rabbitt P.C. On the origins and functions of the enzymes of the 4-chlorobenzoate to 4-hydroxybenzoate converting pathway.Biodegradation. 1994; 5: 259-276Google Scholar); bioluminescence (25Ferri S.R. Meighen E.A. A lux-specific myristoyl transferase in luminescent bacteria related to eukaryotic serine esterase.J. Biol. Chem. 1991; 266: 12852-12857Google Scholar); biosynthesis of polyketides (26Donadio S. Staver M.J. McAlpine J.B. Swanson S.J. Katz L. Modular organization of genes required for complex polyketide biosynthesis.Science. 1991; 252: 675-679Google Scholar); and cleavage of the acyl group from palmitoyl-protein including H-Ras and certain GTP binding proteins (27Camp L.A. Hofmann S.L. Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-ras.J. Biol. Chem. 1993; 268: 22566-22574Google Scholar). As a group, these enzymes vary greatly in molecular weight, cellular location, and substrate specificity. Most thioesterases from animal sources and some bacteria are sensitive to serine-directed inhibitors. A number of mammalian thioesterases have been shown to have a conserved active site motif Gly-X-Ser-X-Gly (16Hardie D.G. Dewart K.B. Aitben A. McCarthy A.D. Amino acid sequence around the reactive serine residue of the thioesterase domain of rabbit fatty acid synthase.Biochim. Biophys. Acta. 1985; 828: 380-382Google Scholar, 17Randhawa Z.I. Naggert J. Blacher R.W. Smith S. Amino acid sequencing of the serine active-site region of the medium-chain S-acyl fatty acid synthetase thioester hydrolase from rat mammary gland.Eur. J. Biochem. 1987; 162: 577-581Google Scholar, 18Tai M-H. Chirala S.S. Wakil S.J. Roles of Ser101, Asp236, and His237 in catalysis of thioesterase II and the C-terminal region of the enzyme in its interaction with fatty acid synthase.Proc. Natl. Acad. Sci. USA. 1993; 90: 1852Google Scholar, 19Witkowski A. Naggert J. Witkowska H.E. Randhawa Z.I. Smith S. Utilization of an active serine 101-cysteine mutant to demonstrate the proximity of the catalytic serine 101 and histidine 237 residues in thioesterase II.J. Biol. Chem. 1992; 267: 18488-18492Google Scholar, 25Ferri S.R. Meighen E.A. A lux-specific myristoyl transferase in luminescent bacteria related to eukaryotic serine esterase.J. Biol. Chem. 1991; 266: 12852-12857Google Scholar). The active site serine is believed to act as a nucleophile during thioester hydrolysis. An examination of the amino acid sequence of the bile acid-CoA hydrolase from Eubacterium sp. strain VPI 12708 found no conserved active site motifs typical of these mammalian thioesterases, but there are a number of cysteine and histidine residues in bile acid-CoA hydrolase. Escherichia coli thioesterase I and several other mammalian thioesterases have a conserved Gly-X-His motif near the carboxyl terminal end of these enzymes (28Cho H. Cronan J.E. Escherichia coli thioesterase I, molecular cloning and sequencing of the structural gene and identification as a periplasmic enzyme.J. Biol. Chem. 1993; 268: 9238-9245Google Scholar), but this motif was not found in bile acid-CoA hydrolase. Amino acid sequence data base searches show no significant similarity with other thioesterases. Surprisingly, significant amino acid sequence similarity over the entireprotein was observed with E. coli carnitine dehydratase (Fig. 6). It is unknown whether carnitine dehydratase has thioesterase activity and to date there is no reported data that might indicate an evolutionary relationship between thioesterases and dehydratases. However, the genes encoding bile acid-CoA hydrolase and carnitine dehydratase may have been derived from a common ancestral gene. 2-Arylpropionyl-CoA epimerase, an important enzyme in ibuprofen metabolism (29Reichel C.R. Brugger R. Bang H. Geisslinger G. Brune K. Molecular cloning and expression of a 2-arylpropionyl-coenzyme A epimerase: a key enzyme in the inversion metabolism of ibuprofen.Mol. Pharmacol. 1997; 51: 576-582Google Scholar), also has significant homology with bile acid CoA hydrolase. The normal physiological role of 2-arylpropionyl CoA epimerase in cell metabolism is unknown. The precise role of bile acid-CoA hydrolase in bile acid 7α-dehydroxylation in Eubacterium sp. strain VPI 12708 is unknown. We have assayed for the ability of bile acid-CoA hydrolase to transfer the CoA moiety of cholyl-CoA to either acetate or cellular proteins, which would conserve the high energy thioester bond. These experiments yielded negative results, but we cannot totally exclude the possibility that this enzyme has CoA transferase activity. Our laboratory has shown that the bai operon of this bacterium encodes a bile acid-CoA ligase which is believed to be the first enzymatic step in the bile acid 7α-dehydroxylation pathway (7Mallonee D.H. Adams J.L. Hylemon P.B. The bile acid-inducible baiB gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A ligase.J. Bacteriol. 1992; 174: 2065-2071Google Scholar). It is unclear whether bile acids remain linked to CoA during all the various reactions of the 7α-dehydroxylation pathway. Nevertheless, CoA conjugates other than cholyl-CoA have been detected in cell extracts of this bacterium and 3α-hydroxysteroid dehydrogenase (baiA) appears to strongly prefer bile acid-CoA conjugates as substrates (8Mallonee D.H. Lijewski M.A. Hylemon P.B. Expression in Escherichia coli and characterization of a bile acid-inducible 3α-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.Curr. Microbiol. 1995; 30: 259-263Google Scholar). The baiE gene has been reported to encode a bile acid 7α-dehydratase. This enzyme does not require bile acid-CoA conjugates as substrates (9Dawson J.A. Mallonee D.H. Björkhem I. Hylemon P.B. Expression and characterization of a C24 bile acid 7α-dehydratase from Eubacterium sp. strain VPI 12708 in Escherichia coli.J. Lipid Res. 1996; 37: 1258-1267Google Scholar). Therefore, the physiological substrate for the bile acid-CoA hydrolase may be 7α,12α-dihydroxy-3-oxo-4-cholenoyl-CoA. The high Km (175 μm) of bile acid-CoA hydrolase for cholyl-CoA suggests that other bile acid intermediates may be the preferred substrates. This would prevent the possibility of a futile cycle between cholic acid-CoA ligase and cholyl-CoA hydrolase. This work was supported by grant PO1-DK38030 from the National Institutes of Health and by a grant from the Swedish Medical Research Council. We would like to thank Dr. Stefan Alexson for his helpful comments on writing the manuscript. bile acid inducible coenzyme A thin-layer chromatography high performance liquid chromatography sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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