Original Article

The bile acid-inducible bai F gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A hydrolase

Correspondence to: Phillip B. Hylemon

	ABSTRACT

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The human intestinal Eubacterium sp. strain VPI 12708 has been shown to have a multistep biochemical pathway for bile acid 7-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 7-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 K _m and V_max 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.

Supplementary key words: 7-dehydroxylation, secondary bile acid

	INTRODUCTION

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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 (1). 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 7-dehydroxylation of cholic acid and chenodeoxycholic acid yielding deoxycholic and lithocholic acid, respectively (2) (3). 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 (4).

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 7-dehydroxylation pathway (5) ( Figure 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 (6), followed by synthesis of the coenzyme A (CoA) conjugate (7), oxidation of the 3-hydroxy group (8), and insertion of a double bond between C-4 and C-5 (5). The next step in this pathway involves 7-dehydration (9), yielding a 3-oxo-^4,6- intermediate that is sequentially reduced in three steps to the 7-dehydroxylated bile acid and released from the cell (Figure 1). It is not known when the coenzyme A is cleaved from the bile acid. However, the 3-hydroxysteroid dehydrogenase, an enzyme in the oxidative arm of the pathway, has been shown to strongly prefer bile acid-CoA conjugates as substrates (8).

View larger version (44K): [in this window] [in a new window]   Figure 1. Proposed cholic acid 7-dehydroxylation pathway in Eubacterium sp. strain VPI 12708. Bile acid intermediates include: 3,7,12-trihydroxy-5ß-cholanoic acid, A; 3,7,12-trihydroxy-5ß-cholyl-CoA, B; 7,12-dihydroxy-3-oxo-5ß-cholyl-CoA, C; 7,12-dihydroxy-3-oxo-4-cholenyl-CoA, D; 7,12-dihydoxy-3-oxo-4-cholenoic acid, E; 12-hydroxy-3-oxo-4,6-choldienoic acid, F; 12-hydroxy-3-oxo-4-cholenoic acid, G; 12-hydroxy-3-oxo-5ß-cholanoic acid, H; and 3,12-dihydroxy-5ß-cholanoic acid, I. The proposed function of the bai gene products are indicated.

A large (~12 kb) bile acid-inducible operon has been cloned and sequenced from Eubacterium sp. strain VPI 12708 (10). 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 7-dehydroxylation pathway has been elucidated ( Figure 2). The baiB gene has been shown to encode a bile acid-CoA ligase (7), 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.

View larger version (12K): [in this window] [in a new window]   Figure 2. The bai operon from Eubacterium sp. strain VPI 12708. The function of various bai gene products are indicted. Abb. NADH:FOR, NADH:flavin oxidoreductase, 3-HSDH; 3-hydroxysteroid dehydrogenase; transporter, bile acid transporter.

	MATERIALS AND METHODS

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Chemicals, enzymes, and bile acid-CoA hydrolase substrates
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 (7). [24-¹⁴C]-3-dehydrocholyl-CoA was synthesized enzymatically from [24-¹⁴C]cholyl-CoA using 3-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).

Chemical synthesis of unlabeled cholyl-CoA
The cholyl-CoA ester was synthesized by the mixed anhydride procedure essentially as described by Shah and Staple (11). 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.

Bacterial strains
E. coli DH5 was obtained from Gibco BRL (Gaithersburg, MD) and used as the host strain for expression of recombinant plasmids.

Expression of the baiF gene in Escherichia coli
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 gt11 vector, and then subcloned into a pUC8 vector (12). The sequence at the 3' end of the baiF gene was obtained through direct sequencing of DNA obtained by inverse PCR (10). 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 Sal I 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 Sal I and BamHI, ligated to pUC19 vector DNA, and transformed into E. coli DH5. 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 (12).

Bile acid-CoA hydrolase assay
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 K_m and V_max were determined using a Hanes plot (13).

Reversed-phase HPLC of reaction products
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.

TLC of bile acid reaction products
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 (14). 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.

Purification of bile acid-CoA hydrolase
Escherichia coli DH5 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.

Analysis of amino acid sequence data
Amino acid sequence analysis and data base searching was performed with the Wisconsin Package (Genetics Computer Group, Madison, WI).

	RESULTS

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Expression of the baiF gene in E. coli
Transformation of the pUC19-47K plasmid into E. coli resulted in excellent expression and accumulation of the baiF gene product ( Figure 3, compare lanes 1 and 2). Cell extracts of E. coli DH5 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 ( Figure 4).

View larger version (41K): [in this window] [in a new window]   Figure 3. Purification of bile acid-CoA hydrolase from E. coli expressing the baiF gene. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude and pooled fractions from various purification steps stained with Coomassie blue. Molecular weight standards are indicated on the left; 1. E. coli DH5 without plasmid construct (25 µg); 2. E. coli DH5 containing pUC19-47K plasmid (25 µg); 3. pooled DEAE fractions (8 µg); 4. Pooled Mono-Q fraction (2.5 µg).

View larger version (27K): [in this window] [in a new window]   Figure 4. Western immunoblot of baiF protein. Two µg of total soluble protein was used per lane for Eubacterium sp. strain VPI 12708 (1), E. coli DH5 pUC19-47K (2), and E. coli DH5 control (3).

Purification of bile acid-CoA hydrolase
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 (Figure 3). The final enzyme preparation was greater than 90% pure (Figure 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 when stored at -20°C in 50% glycerol. E. coli DH5 (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).

Optimization of bile acid-CoA hydrolase activity
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.

Determination of apparent kinetic constants
K_m and V_vax values were obtained using initial velocity conditions. The approximate K_m and V_max values for cholyl-CoA hydrolysis were 175 µM and 374 µmol/min per mg protein, respectively. Kinetic constants were determined using a Hanes Plot (13).

Bile acid-CoA hydrolase substrate specificity and product identification
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 ( Figure 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.

View larger version (20K): [in this window] [in a new window]   Figure 5. Elution profiles of bile acid-CoA hydrolase product from C-18 reversed-phase HPLC using a 5–40% isopropanol gradient in 20 mM ammonium bicarbonate buffer (described in Materials and Methods). Shown are elution profiles of bile acid-CoA hydrolase products at 0, 2, and 10 min after enzyme addition to reaction mixtures. The elution times from coenzyme A and cholyl-CoA are indicated.

Amino acid sequence analysis of bile acid-CoA hydrolase
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 (15) and Figure 6. However, the baiF gene product had no bile acid 7-dehydratase activity using 7,12-dihydroxy-3-oxo-4-cholenoic acid as substrate (data not shown). No sequence homology was found with any reported 3-ketoacyl-CoA thiolases, acyl-CoA hydrolases, or thiotransferases. In addition, consensus active site amino acid sequences for serine esterases (16) (17) (18) (19), CoA transferases/acyl-CoA hydrolases (20), and 3-ketoacyl-CoA thiolases (21) could not be found in bile acid coenzyme A hydrolase.

View larger version (49K): [in this window] [in a new window]   Figure 6. Amino acid sequence alignment of bile acid-coenzyme A hydrolase from Eubacterium sp. strain VPI 12708 and carnitine dehydratase from E. coli using the GCG gap program. Vertical lines and colons indicate amino acid sequence identity and similarity, respectively.

	DISCUSSION

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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 (22); hydrolysis of fatty acids from acyl-carrier protein (23); dehalogenation of chlorinated aromatic compounds in bacteria (24); bioluminescence (25); biosynthesis of polyketides (26); and cleavage of the acyl group from palmitoyl-protein including H-Ras and certain GTP binding proteins (27). 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 (16) (17) (18) (19) (25). 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 (28), 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 entire protein was observed with E. coli carnitine dehydratase (Figure 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 (29), 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 7-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 7-dehydroxylation pathway (7). It is unclear whether bile acids remain linked to CoA during all the various reactions of the 7-dehydroxylation pathway. Nevertheless, CoA conjugates other than cholyl-CoA have been detected in cell extracts of this bacterium and 3-hydroxysteroid dehydrogenase (baiA) appears to strongly prefer bile acid-CoA conjugates as substrates (8). The baiE gene has been reported to encode a bile acid 7-dehydratase. This enzyme does not require bile acid-CoA conjugates as substrates (9). Therefore, the physiological substrate for the bile acid-CoA hydrolase may be 7,12-dihydroxy-3-oxo-4-cholenoyl-CoA. The high K_m (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.

	ACKNOWLEDGMENTS

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.

Manuscript received May 21, 1998; and in revised form August 21, 1998.

Abbreviations: bai, bile acid inducible; CoA, coenzyme A; TLC, thin- layer chromatography; HPLC, high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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