Identification and characterization of two bile acid coenzyme A transferases from Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium
2011; Elsevier BV; Volume: 53; Issue: 1 Linguagem: Inglês
10.1194/jlr.m020313
ISSN1539-7262
AutoresJason M. Ridlon, Phillip B. Hylemon,
Tópico(s)Folate and B Vitamins Research
ResumoThe human bile acid pool composition is composed of both primary bile acids (cholic acid and chenodeoxycholic acid) and secondary bile acids (deoxycholic acid and lithocholic acid). Secondary bile acids are formed by the 7α-dehydroxylation of primary bile acids carried out by intestinal anaerobic bacteria. We have previously described a multistep biochemical pathway in Clostridium scindens that is responsible for bile acid 7α-dehydroxylation. We have identified a large (12 kb) bile acid inducible (bai) operon in this bacterium that encodes eight genes involved in bile acid 7α-dehydroxylation. However, the function of the baiF gene product in this operon has not been elucidated. In the current study, we cloned and expressed the baiF gene in E. coli and discovered it has bile acid CoA transferase activity. In addition, we discovered a second bai operon encoding three genes. The baiK gene in this operon was expressed in E. coli and found to encode a second bile acid CoA transferase. Both bile acid CoA transferases were determined to be members of the type III family by amino acid sequence comparisons. Both bile acid CoA transferases had broad substrate specificity, except the baiK gene product, which failed to use lithocholyl-CoA as a CoA donor. Primary bile acids are ligated to CoA via an ATP-dependent mechanism during the initial steps of 7α-dehydroxylation. The bile acid CoA transferases conserve the thioester bond energy, saving the cell ATP molecules during bile acid 7α-dehydroxylation. ATP-dependent CoA ligation is likely quickly supplanted by ATP-independent CoA transfer. The human bile acid pool composition is composed of both primary bile acids (cholic acid and chenodeoxycholic acid) and secondary bile acids (deoxycholic acid and lithocholic acid). Secondary bile acids are formed by the 7α-dehydroxylation of primary bile acids carried out by intestinal anaerobic bacteria. We have previously described a multistep biochemical pathway in Clostridium scindens that is responsible for bile acid 7α-dehydroxylation. We have identified a large (12 kb) bile acid inducible (bai) operon in this bacterium that encodes eight genes involved in bile acid 7α-dehydroxylation. However, the function of the baiF gene product in this operon has not been elucidated. In the current study, we cloned and expressed the baiF gene in E. coli and discovered it has bile acid CoA transferase activity. In addition, we discovered a second bai operon encoding three genes. The baiK gene in this operon was expressed in E. coli and found to encode a second bile acid CoA transferase. Both bile acid CoA transferases were determined to be members of the type III family by amino acid sequence comparisons. Both bile acid CoA transferases had broad substrate specificity, except the baiK gene product, which failed to use lithocholyl-CoA as a CoA donor. Primary bile acids are ligated to CoA via an ATP-dependent mechanism during the initial steps of 7α-dehydroxylation. The bile acid CoA transferases conserve the thioester bond energy, saving the cell ATP molecules during bile acid 7α-dehydroxylation. ATP-dependent CoA ligation is likely quickly supplanted by ATP-independent CoA transfer. The human liver synthesizes two primary bile acids from cholesterol, chenodeoxycholic acid (CDCA; 3α, 7α-dihydro-5β-cholan-24-oic acid) and cholic acid (CA; 3α, 7α, 12α-trihydroxy-5β-cholan-24-oic acid). Bile acids are conjugated to either taurine or glycine before active secretion into bile and in this form are termed bile salts. Bile salts function to solubilize lipids and lipid soluble vitamins from the duodenum through the jejunum of the small intestine. Upon reaching the terminal ileum, bile salts are actively transported across the intestinal epithelium into the portal blood and returned to the liver. This process is termed enterohepatic circulation and is ∼95% efficient. However, roughly 400-600 mg of bile acids enter the large intestine every day, where they are metabolized by a diversity of microorganisms (1.Ridlon J.M. Kang D. Hylemon P.B. Bile salt biotransformations by Human Intestinal bacteria.J. Lipid Res. 2006; 47: 241-259Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar). Bile salts are rapidly deconjugated and converted to the secondary bile acids, deoxycholic acid (3α,12α-trihydroxy-5β-cholanoic acid) and lithocholic acid (3α-hydroxy-5β-cholan-24-oic acid) from CA and CDCA, respectively. Decades of research strongly suggest that secondary bile acids are involved in human disease processes, including cancers of the colon, esophagus, and biliary track (2.Bernstein H. Bernstein C. Payne C.M. Dvorakova K. Garewal H. Bile acids as carcinogens in human gastrointestinal cancers.Mutat. Res. 2005; 589: 47-65Crossref PubMed Scopus (451) Google Scholar), as well as cholesterol gallstone disease in some patients with high levels of deoxycholic acid in bile (3.Berr F. Kullak-Ublick G.A. Paumgartner G. Munzig W. Hylemon P.B. 7alpha-dehydroxylating bacteria enhance deoxycholic acid input and cholesterol saturation of bile in patients with gallstones.Gastroenterology. 1996; 111: 1611-1620Abstract Full Text PDF PubMed Scopus (117) Google Scholar). The level of deoxycholic acid in bile is believed to be controlled by two major factors: i) levels and activities of bile acid 7α-dehydroxylating gut bacteria (3.Berr F. Kullak-Ublick G.A. Paumgartner G. Munzig W. Hylemon P.B. 7alpha-dehydroxylating bacteria enhance deoxycholic acid input and cholesterol saturation of bile in patients with gallstones.Gastroenterology. 1996; 111: 1611-1620Abstract Full Text PDF PubMed Scopus (117) Google Scholar) and ii) colonic transit time (4.Dowling R.H. Veysey M.J. Pereira S.P. Hussaini S.H. Thomas L.A. Wass J.A. Murphy G.M. Role of intestinal transit in the pathogenesis of gallbladder stones.Can. J. Gastroenterol. 1997; 11: 57-64Crossref PubMed Scopus (37) Google Scholar). Colonic pH may also be a minor factor. Therefore, an understanding of the genetics and enzymology of bile acid 7α-dehydroxylation is an important first step in finding ways to decrease secondary bile acids with potentially beneficial outcomes to the host. Several bacterial species in the genus Clostridium have been isolated and shown to convert primary bile acids into secondary bile acids, a process termed bile acid 7α-dehydroxylation (1.Ridlon J.M. Kang D. Hylemon P.B. Bile salt biotransformations by Human Intestinal bacteria.J. Lipid Res. 2006; 47: 241-259Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar). We previously proposed a multistep biochemical pathway for bile acid 7α-dehydroxylation (Fig. 1). In addition, we characterized the bile acid-inducible (bai) operons from C. scindens VPI 12708 (5.Mallonee 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: 7011-7019Crossref PubMed Scopus (69) Google Scholar), C. hiranonis DSM13275 (6.Wells J.E. Hylemon P.B. Identification and characterization of a bile acid 7α-dehydroxylation operon in Clostridium sp. strain TO-931, a highly active 7α-dehydroxylating strain isolated from human feces.Appl. Environ. Microbiol. 2000; 66: 1107-1113Crossref PubMed Scopus (111) Google Scholar), and C. hylemonae DSM 15053 (7.Ridlon J.M. Kang D. Hylemon P.B. Isolation and characterization of a bile acid inducible 7alpha-dehydroxylating operon in Clostridium hylemonae DSM 15053.Anaerobe. 2010; 16: 137-146Crossref PubMed Scopus (79) Google Scholar) that encode enzymes involved in the initial steps of the bile acid 7α-dehydroxylation pathway (Fig. 2). The current model of bile acid 7α-dehydroxylation suggests that free primary bile acids are actively transported into the bacterial cell by a proton-dependent bile acid transporter encoded by the baiG gene (8.Mallonee 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: 7053-7058Crossref PubMed Google Scholar). Once inside, the primary bile acid is ligated to CoA in an ATP-dependent manner by the baiB gene product (9.Mallonee 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: 2065-2071Crossref PubMed Google Scholar). The baiA gene encodes a bile acid, 3α-hydroxysteroid dehydrogenase, specific for primary bile acid CoA conjugates (10.Mallonee D.H. Lijewski M.A. Hylemon P.B. Expression in Escherichia coli and characterization of a bile acidinducible 3α-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.Curr. Microbiol. 1995; 30: 259-263Crossref PubMed Scopus (33) Google Scholar). We recently reported that the baiCD and baiH gene products encode stereospecific 3-dehydro-4-bile acid oxidoreductases recognizing 7α-hydroxy bile acids (CA, CDCA) and 7β-hydroxy bile acids [ursodeoxycholic acid (UDCA),; 3α, 7α-dihydro-5β-cholan-24-oic acid], respectively (11.Kang D. Ridlon J.M. Moore 2nd, D.R. Barnes S. Hylemon P.B. Clostridium scindens baiCD and baiH genes encode stereo-specific 7alpha/7beta-hydroxy-3-oxo-delta4-cholenoic acid oxidoreductases.Biochim. Biophys. Acta. 2008; 1781: 16-25Crossref PubMed Scopus (58) Google Scholar). The rate-limiting and irreversible step in this pathway is 7α-dehydration. This reaction is catalyzed by bile acid 7α-dehydratase, which is encoded by the baiE gene (12.Dawson J.A. Mallonee D.H. Björkhem I. Hylemon P.B. Expression and characterization of a C-24 bile acid 7α-dehydratase from Eubacterium sp. strain VPI 12708 in Escherichia coli.J. Lipid Res. 1996; 37: 1258-1267Abstract Full Text PDF PubMed Google Scholar). The 3-dehydro-4,6-bile acid intermediate is then sequentially reduced and exported from the cell; however, genes in the "reductive arm" of the pathway have yet to be identified. Previously, we showed that the baiF gene product hydrolyzes bile acid CoA conjugates (13.Ye H.Q. Mallonee D.H. Wells J.E. Björkhem I. Hylemon P.B. The bile acid-inducible baiF gene from Eubacterium sp. strain VPI 12708 encodes a bile acid-coenzyme A hydrolase.J. Lipid Res. 1999; 40: 17-23Abstract Full Text Full Text PDF PubMed Google Scholar). However, others and we have hypothesized, based on amino acid sequence comparisons with known CoA transferases, that this gene product may be a bile acid CoA transferase (1.Ridlon J.M. Kang D. Hylemon P.B. Bile salt biotransformations by Human Intestinal bacteria.J. Lipid Res. 2006; 47: 241-259Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 14.Heider J. A new family of CoA-transferases.FEBS Lett. 2001; 509: 345-349Crossref PubMed Scopus (109) Google Scholar). Here, we present strong evidence that the baiF gene encodes a bile acid CoA transferase with broad bile acid substrate specificity. In addition, we report the discovery and characterization of a second bile acid CoA transferase encoded on what appears to be second multigene operon involved in bile acid metabolism in C. scindens.Fig. 2Schematic representation of bile acid 7α-dehydroxylation operons from C. scindens VPI 12708 and C. hylemonae DSM 15053. "P" represents putative promoter regions.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Clostridium scindens ATCC 35704 was purchased from American Type Culture Collection. C. scindens VPI 12708 was stored as a 30% glycerol stock at −80°C prior to this study, and working stocks were cultivated in chopped meat broth. E. coli BL21 (DE3) RIL was purchased from Agilent Stratagene (Santa Clara, CA). E. coli was cultivated in Luria-Bertani medium containing appropriate antibiotics. C. scindens strains were cultivated in brain/heart infusion broth under nitrogen. Bile acids were purchased from Sigma-Aldrich (St. Louis, MO) and Steraloids (Newport, RI). ADCA was a generous gift from Dr. James P. Coleman, East Carolina University. CoA was purchased from Sigma-Aldrich. Genomic DNA was isolated from C. hylemonae, C. scindens VPI 12708, and C. scindens ATCC 35704 by enzymatic treatment, followed by bead beating as described previously (7.Ridlon J.M. Kang D. Hylemon P.B. Isolation and characterization of a bile acid inducible 7alpha-dehydroxylating operon in Clostridium hylemonae DSM 15053.Anaerobe. 2010; 16: 137-146Crossref PubMed Scopus (79) Google Scholar). Genome-walking DNA libraries were prepared using the Universal GenomeWalker Kit (Clontech) according to the manufacturer. The DNA extraction method used in this application and PCR conditions have been described previously (7.Ridlon J.M. Kang D. Hylemon P.B. Isolation and characterization of a bile acid inducible 7alpha-dehydroxylating operon in Clostridium hylemonae DSM 15053.Anaerobe. 2010; 16: 137-146Crossref PubMed Scopus (79) Google Scholar). Total RNA was isolated as described previously (7.Ridlon J.M. Kang D. Hylemon P.B. Isolation and characterization of a bile acid inducible 7alpha-dehydroxylating operon in Clostridium hylemonae DSM 15053.Anaerobe. 2010; 16: 137-146Crossref PubMed Scopus (79) Google Scholar). cDNA for reverse transcriptase PCR was generated with the Advantage RT-for-PCR kit (Clontech). cDNA for determination of transcription initiation site was generated using the SMART-RACE cDNA synthesis kit (Clontech) according to the manufacturer, with the exception that a gene-specific primer replaced 5′ CDS primer. The gene-specific primer for determination of the baiJ gene transcription initiation site was SR12708J 5′-TTC TAT GTC CTC ATC CGT TGC CCC TG-3′. The gene-specific primer for determination of the baiJ gene transcription initiation site was DSM 15053JRace1 5′-TGC GGT AGA GCC TCC TGT GAT ACC TTG-3′. PCR was performed using the Advantage-GC 2 PCR Kit (Clontech) according to the manufacturer. An amount of 0.5 μl GC-Melt was added to each 50 μl reaction. PCR products were excised from agarose gels, GENECLEAN purified, cloned into a pCR 8 GW TOPO TA vector, and sequenced at the Virginia Commonwealth University Nucleic Acid Research Facility. The baiF gene insert was amplified with forward primer 5′-CTGCAGAGAAAAGGAGGTATAAAAACATGGCTGG-3′ (PstI site in italics) and reverse primer 5′GGATCCTTATTTTTCGAACTGCGGGTGGCTCCACTCCTCTTTCTTTCTCATATGT-3′ (BamHI site in italics). The baiK gene insert was amplified with forward primer 5′-CTG CAG GGA AAG GAG GTT ATA GGA ATG AAA GG-3′ (PstI site in italics) and reverse 5′-GGA TCC TTA TTT TTC GAA CTG CGG GTG GCT CCA TTC TTC CTC CTT TTT CCA ACG TTC-3′ (BamHI site in italics). The streptavidin tag sequence (bold) was incorporated into the C terminus of bile acid CoA transferases for one-step purification (15.Schmidt T.G.M. Skerra A. The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins.Nat. Protoc. 2007; 2: 1528-1535Crossref PubMed Scopus (454) Google Scholar). PCR products were cloned into a pCR8GW TOPO TA vector (Invitrogen) and transformed by heat shock at 42°C into One Shot Chemically competent E. coli that accompanies the vector. Plasmids were isolated using the Qiaprep Miniprep Kit (Qiagen) and double-digested with PstI and BamHI (New England Biolabs). Expression vector pSport-1 was double-digested with the same restriction enzymes, and both inserts and pSport-1 linear plasmid were gel-purified using the GENECLEAN Spin Kit (MP Biomedicals). Ligation was performed using T4 DNA ligase according to the manufacturer (New England Biolabs). Ligated vector was transformed into E. coli DH5 (NEB 5α; New England Biolabs) and cultivated in LB medium containing 100 μg/ml ampicillin. pSport vectors containing insert (hereafter referred to as pSportbaiFCTSBP and pSportbaiKCTSBP) as determined by double-digestion and agarose gel electrophoresis were sequenced at the Nucleic Acid Core Facility at Virginia Commonwealth University. Expression vectors were transformed into E. coli BL21 (DE3)RIL by heat shock. Colonies were selected and cultivated overnight in 1 ml LB medium containing 100 μg/ml ampicillin (LB Amp). A 5% inoculum was transferred to LB Amp at 37°C and cultivated until reaching an OD600nm 0.4, at which point gene expression was induced with 1 mM IPTG. Overexpression occurred for 3 h, at which point cells were centrifuged and washed with Buffer A (two volumes 20 mM sodium phosphate; 0.1 M sodium chloride; 10 mM 2-mercaptoethanol; 15% glycerol, pH 7.0). Cells were frozen at −20°C until further processing. Cell lysates were produced by resuspending cells in Buffer A containing 700 μg lysozyme and incubating on ice for 1 h followed by three rounds of sonication (8 s burst, 20 s pause, 2 min total). Benzonase (25 U; Novagen) was added to degrade nucleic acids. Crude lysate was centrifuged 30 min at 16,000 g. Lysate was loaded onto Streptactin Superflow Resin (IBA) equilibrated with Buffer A and washed according to the manufacturer instructions. Elution was performed with Buffer A containing 2.5 μM desthiobiotin. Protein was concentrated on Amicon Ultracel-50K centrifugal filters (Millipore, Cork, Ireland) and stored in 50% glycerol at −20°C. Protein was stable for up to 1 month without detectable loss of activity. Protein samples were separated by 12% SDS-PAGE and transferred to PVDF Western blotting membranes (Roche). Expression and identity of the recombinant streptavidin-tagged proteins was verified using Strep Tag II antibody HRP conjugate (Novagen). CoA conjugates of ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), cholic acid (CA), allocholic acid (ACA), DCA, allodeoxycholic acid (ADCA), lithocholic acid (LCA), and β-muricholic acid (β-MCA) were synthesized according to the method of Killenberg and Dukes (16.Killenberg P.G. Dukes D.F. Coenzyme A derivatives of bile acids-chemical synthesis, purification, and utilization in enzymic preparation of taurine conjugates.J. Lipid Res. 1976; 17: 451-455Abstract Full Text PDF PubMed Google Scholar), with the exception that free bile acids were extracted with ethyl acetate rather than ether. ADCA-CoA synthesized from hydrolyzed rabbit gallstones was separated from contaminating DCA-CoA by reverse-phase HPLC on an HP 1100 series equipped with a Coulter Ultrasphere semipreparative column (5 µm silica gel) (10 mm × 25 cm). Samples (100 µl) were loaded on the column (40°C) and eluted with 5-40% isopropanol in 20 mM ammonium acetate buffer (pH 7.0) over a 40 min period with a flow rate of 2 ml/min. Free CoA and bile acid CoA conjugates were monitored at 260 nm. Isopropanol from fractions containing ADCA-CoA conjugates was evaporated under nitrogen and purified by Sep Pak C-18 chromatography. Columns were washed and equilibrated with 10 ml methanol followed by 10 ml deionized water and Buffer B (3 ml 20 mM sodium phosphate buffer; 0.1 M NaCl, pH 6.0). Bile acid CoA conjugates were dissolved in Buffer B and loaded onto the column, followed by washing with 10 ml water and elution with 2 ml absolute methanol. Samples were concentrated under nitrogen and resuspended in 50% methanol. Concentrated bile acid CoA conjugates were resuspended in 0.1 M sodium acetate buffer (pH 5.5) and stored at −20°C. Concentrations were determined by UV spectrophotometry at A260 nm using a published millimolar extinction coefficient of 15.03 (16.Killenberg P.G. Dukes D.F. Coenzyme A derivatives of bile acids-chemical synthesis, purification, and utilization in enzymic preparation of taurine conjugates.J. Lipid Res. 1976; 17: 451-455Abstract Full Text PDF PubMed Google Scholar). An HPLC assay was used to measure CoA transferase activity. Assays contained 0.01 μg enzyme of recombinant BaiF-SBP or 0.03 μg recombinant BaiK-SBP and 300-500 μM secondary bile acid CoA conjugate with an equal amount of free bile acid acceptor. Reactions were performed at 37°C and terminated at 1 min by addition of 75 μl 1N HCl. Samples were prepared for HPLC by Sep Pak C-18 chromatography as described above. Concentrated reaction products were resuspended in 125 μl 50% methanol. Samples (100 μl) were autoinjected onto an Agilent 1200 series HPLC equipped with an Agilent Eclipse XDB-C18 column (5 μm, 4.6 × 250 mm). CoA conjugates were separated in a buffer system of 20 mM ammonium acetate buffer (pH 6.0) with a linear gradient 0-55% isopropanol over a 35 min period. CoA conjugates were detected at 260 nm and concentrations determined by comparison of peak areas versus A260 nm against concentration curves of known quantity. Assays testing transfer of CoA from DCA-CoA or from ADCA-CoA to CDCA required measurement of the production of [24-14C]CDCA-CoA by liquid scintillation spectrometry instead of reverse-phase HPLC separation because nearly identical hydrophobicities result in comigration of substrate and product. Following termination of reaction by HCl, free [24-14C]CDCA was extracted from the reaction mixture by organic extraction twice with two volumes ethyl acetate. Radioactive counts in the aqueous phase thus measure [24-14C]CDCA-CoA, which is not soluble in ethyl acetate. Samples were separated by reverse-phase HPLC, and fractions corresponding to peaks absorbing at 260 nm were collected and counted by liquid scintillation spectrometry. Values were normalized relative to fractions collected from reactions incubated with [24-14C] CA. One unit of bile acid CoA transferase activity is defined as the amount of enzyme required to produce 1 μmole of primary bile acid CoA conjugate formed per minute. Nucleotide and amino acid sequence alignments were performed using ClustalW (17.Thompson J.D. Higgins D.G. Gibson T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55638) Google Scholar). Amino acid sequence alignments were represented using Boxshade. Protein Mr calculations were performed using the Compute pI/MW tool on the ExPASy Proteomics Server. Open reading frames (ORF) were detected using the ORF Finder Program at the NCBI website and searched against GenBank by BLAST. GenBank accession numbers for the baiJKL operon from C. hylemonae DSM 15053 is EU675331, and baiJKL operon from C. scindens VPI 12708 is EU675330. We previously reported multiple copies of the baiA gene, which encodes a primary bile acid, CoA-dependent 3α-HSDH, in the chromosome of C. scindens VPI 12708 (18.Gopal-Srivastava R. Mallonee D.H. White W.B. Hylemon P.B. Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.J. Bacteriol. 1990; 172: 4420-4426Crossref PubMed Google Scholar). One copy of the baiA gene is located within in the large bai operon, and the other is a single cholic acid-inducible gene at a different chromosomal location (Fig. 2). More recently, we have been able to isolate and identify the baiA genes in C. hylemonae (7.Ridlon J.M. Kang D. Hylemon P.B. Isolation and characterization of a bile acid inducible 7alpha-dehydroxylating operon in Clostridium hylemonae DSM 15053.Anaerobe. 2010; 16: 137-146Crossref PubMed Scopus (79) Google Scholar). Subsequent upstream DNA sequencing from the single baiA gene in this bacterium revealed a cluster of genes that appeared to be involved in bile acid metabolism by amino acid sequence analysis (Fig. 3). These same genes were also found in C. scindens VPI 12708 but not in C. scindens ATCC 35704, for which a total genome DNA sequence is available. An ORF (baiL) upstream of the baiA gene in C. hylemonae is predicted to encode a short chain pyridine nucleotide-dependent alcohol/polyol oxidoreductase. Members of this family include the baiA gene. However, these two gene products shared only 19% amino acid sequence identity and 35% similarity. A second ORF (baiK) was located upstream of the baiL gene, and amino acid sequence analysis suggests it may encode a type III CoA transferase. It has very high amino acid sequence identity with the baiF gene product from C. scindens VPI 12708 (63% identity; 77% similarity), as well as other type III CoA transferases reported in the literature (Fig. 4). Directly upstream from the hypothetical CoA transferase gene was a third ORF (baiJ) encoding a putative flavoprotein. BLAST analysis suggested this gene product encodes a flavoprotein similar to fumarate reductases and 3-ketosteroid-Δ1-dehydrogenases. The former can likely be ruled out due to the lack of four conserved N-terminal heme binding sites (CXXCH) found in fumarate reductases, such as that of Shewanella putrefaciens MR-1, whose structure has been solved (19.Leys D. Tsapin A.S. Nealson K.H. Meyer T.E. Cusanovich M.A. Van Beeumen J.J. Structure and mechanism of the flavocytochrome c fumarate reductase of Shewanella putrefaciens MR-1.Nat. Struct. Biol. 1999; 6: 1113-1117Crossref PubMed Scopus (133) Google Scholar). These three gene products share 89% amino acid sequence identity between C. scindens VPI 12708 and C. hylemonae DSM 15053.Fig. 4Boxshade alignment of baiF and baiK gene products with members of type III CoA transferase family. fCTOx represents formyl CoA transferase from Oxalobacter formigenes (AAC45298.1), caiBEco represents carnitine CoA transferase from E. coli (BAB96607.1), and YfdwA represents the formyl CoA transferase from E. coli (YP_853505). † Active site Asp169 residue.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition, an ORF upstream of the baiJ gene on the antisense strand encodes a putative member of the LysR family of transcription factors. A second ORF also located on the antisense strand relative to the baiJ gene was found upstream of the putative transcription factor. This ORF was predicted to encode a polypeptide in the tryptophan-rich sensory protein (TspO)/mitochondrial benzodiazepine receptor (MBR) homolog family of signal transduction integral membrane proteins. Farther upstream, and on the same strand relative to the baiJ gene, we located the baiAl gene and promoter region that has been previously reported (18.Gopal-Srivastava R. Mallonee D.H. White W.B. Hylemon P.B. Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.J. Bacteriol. 1990; 172: 4420-4426Crossref PubMed Google Scholar). This gene was located directly upstream of the TspO homolog. Note that the single baiA genes from both C. scindens VPI 12708 and C. hylemonae were found in proximity to this novel bai operon (Fig. 3). Some notable features of this novel operon include the similar organization of the baiJKL genes, upstream conserved elements with the oxidative bai upstream region, and proximity of the transcription initiation site. Interestingly, the upstream ORFs are not conserved between C. scindens and C. hylemonae. Each region includes an ORF predicted to encode a transcription factor of a different family of proteins on opposite strands, which might indicate differences in transcriptional regulation. In addition, the ORF immediately upstream of the baiJ gene from C. hylemonae DSM 15053 is predicted to encode a 4Fe-4S ferrodoxin. This ORF was not located adjacent to the baiJKL genes from C. scindens. The baiJKL genes were searched against total genome sequencing data of C. scindens ATCC 35704 and were not detected. Moreover, these genes were not detected by PCR amplification of the baiK gene using cloning primers (see Materials and Methods). These results suggest that these genes are missing in this strain of bile acid 7α-dehydroxylating intestinal bacteria. We located a sequence 24 bp upstream of thebaiJ ORF in C. hylemonae with conserved elements with that of the regions upstream of the baiBCDAGHI and baiA operons from C. scindens, C. hylemonae, and C. hiranonis. 5′ SMART RACE PCR was performed on the baiJ gene, and the mRNA start site was determined at the 5′ end of the conserved bai region, suggesting the promoter is located upstream of this conserved region (Fig. 5). It is quite possible that this conserved region is a promoter region for both the baiA and baiB operons; however, this region does not appear to be a promoter for the baiJKL genes. The functional significance of this conserved region remains to be elucidated. This region was also located directly upstream of the baiJ gene in C. scindens VPI 12708 and shared a high degree of sequence identity with the putative bai promoter region found upstream of the baiJ gene of C. hylemonae. Finally, cDNA was prepared from CA versus uninduced control cells and primers were designed at the 3′ end of the baiJ gene and the 5′ end of the baiK gene and between baiK and baiL for determination of coexpression. Indeed, the baiJKL genes appear to be expressed as a polycistronic (single mRNA) operon (Fig. 5D). The baiF gene was PCR amplified from purified genomic DNA from Clostridium scindens ATCC 35704 and cloned into a pSport-1 expression vector. The baiF gene was overexpressed in E. coli BL21(DE3)RIL, producing a C-terminal streptavidin-tagged recombinant protein. One-step purification from 300 mg protein yielded 600 μg BaiF-Strep tag fusion protein, which matches 2% total protein expression estimated by SDS-PAGE gel (Fig. 6A). Western blot analysis using Strep Tag II antibody confirmed identity of the recombinant BaiF-Strep tag fusion protein. The baiK gene was also cloned into a pSport-1 expression vector and expressed as a C-terminal streptavidin-tagged recombinant protein. Expression and one-step purification from 300 mg crude extract resulted in recovery of 500 μg purified enzyme. The deduced molecular weight of the BaiK is 49.5 kDa, which was confirmed by SDS-PAGE and Western immunoblotting using Strep-Tag II antibody (Fig. 6B). T
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