Identification and characterization of two bile acid coenzyme A transferases from Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium
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 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 C. scindens VPI 12708 was as a to this study, and were in E. coli was purchased from E. coli was in C. scindens were in Bile acids were purchased from and was a from CoA was purchased from was isolated from C. C. scindens VPI and C. scindens ATCC 35704 by by 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). were the to the The in this and 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). 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). for was with the for of was the to the with the that a The for of the gene was The for of the gene was DSM was the to the of was to products were from cloned into a and the The baiF gene was with in and in The baiK gene was with in and in The sequence was into the of bile acid CoA transferases for The for and or of PubMed Scopus Google Scholar). products were cloned into a and by into E. coli that the were isolated the and with and Expression was with the and both and were the was to the was into E. coli and in to as and as determined by and were the Expression were into E. coli by were and in A was to and reaching an which gene expression was with for which were and with A pH were were by in A and on for by three of was to was was with A and to the was with A was on and in was for to of activity. were by and to Expression and of the was CoA conjugates of acid chenodeoxycholic acid cholic acid acid acid lithocholic acid and acid were to the of and A of bile and in of taurine Lipid Res. 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Full Text PDF PubMed Google Scholar). was to CoA transferase activity. of or and secondary bile acid CoA with an of free bile acid were and by of were for by as described reaction products were in were an with an CoA conjugates were in a of with a a CoA conjugates were and determined by of of known of CoA from or from to of the of by of in of substrate and of reaction by free was from the reaction by with two in the which is not soluble in were by and to were and by were to from with of bile acid CoA transferase is as the of to of primary bile acid CoA formed and amino acid sequence were the of sequence through sequence and Res. PubMed Scopus Google Scholar). acid sequence were were the on the were the the and by for the operon from C. hylemonae DSM 15053 is and operon from C. scindens VPI 12708 is We previously reported of the baiA which encodes a primary bile acid, in the of C. scindens VPI 12708 Mallonee D.H. White W.B. Hylemon P.B. of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.J. Bacteriol. 1990; 172: PubMed Google Scholar). of the baiA gene is in the large bai and the is a cholic acid-inducible gene a (Fig. 2). we have been to and 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). sequencing from the baiA gene in this bacterium a of genes that to be involved in bile acid metabolism by amino acid sequence (Fig. genes were also found in C. scindens VPI 12708 not in C. scindens ATCC for which a sequence is of the baiA gene in C. hylemonae is to encode a of this family the baiA However, two gene products amino acid sequence and A second was of the and amino acid sequence suggests it may encode a type III CoA transferase. has high amino acid sequence with the baiF gene product from C. scindens VPI 12708 as well as type III CoA transferases reported in the (Fig. from the CoA transferase gene was a encoding a putative this gene product encodes a to and The likely be out to the of found in as that of has been D. M.A. and mechanism of the of 1999; PubMed Scopus Google Scholar). three gene products amino acid sequence C. scindens VPI 12708 and C. hylemonae DSM of baiF and baiK gene products with members of type III CoA transferase represents CoA transferase from represents CoA transferase from E. coli and represents the CoA transferase from E. coli Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition, an of the gene on the encodes a putative of the family of A second also on the to the gene was found of the putative factor. This was to encode a in the family of and on the to the we the gene and promoter that has been previously reported Mallonee D.H. White W.B. Hylemon P.B. of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.J. Bacteriol. 1990; 172: PubMed Google Scholar). This gene was of the that the baiA genes from both C. scindens VPI 12708 and C. hylemonae were found in to this bai operon (Fig. of this operon the of the with the bai and of the the are not C. scindens and C. an to encode a of a family of on which in In addition, the of the gene from C. hylemonae DSM 15053 is to encode a This was not to the genes from C. The genes were sequencing of C. scindens ATCC 35704 and were not genes were not by of the baiK gene and suggest that genes are in this strain of bile acid 7α-dehydroxylating intestinal bacteria. We a sequence of in C. hylemonae with with that of the of the and baiA operons from C. C. and C. was on the and the was determined the of the bai the promoter is of this (Fig. is that this is a promoter for both the baiA and baiB however, this not to be a promoter for the genes. The of this to be elucidated. This was also of the gene in C. scindens VPI 12708 and a high of sequence with the putative bai promoter found of the gene of C. was from CA and were the of the gene and the of the baiK gene and baiK and for of the genes to be expressed as a operon (Fig. The baiF gene was from from Clostridium scindens ATCC 35704 and cloned into a expression The baiF gene was in E. coli a from mg which expression by (Fig. of the The baiK gene was also cloned into a expression and expressed as a Expression and from mg in of The of the is which was by and (Fig. The in CoA bile acids In addition, secondary bile conjugates are with from primary bile conjugates by We that bile acid CoA from secondary bile acid CoA conjugates to free primary bile acids (Fig. 1). We the of primary bile acid CoA conjugates by by saturation were for the baiF and baiK gene products with and with CA as CoA not substrate to be to a were of and with of primary bile acid in not to be a substrate for the baiK gene product 1). The reaction was a and and of bile acid CoA of and were of is as the of to CoA from to CA of and were of is as the of to CoA from to CA and in as with reaction not not of and were of is as the of to CoA from to CA in a new in as with reaction not not We determined that on of the to a reaction and CA in a product (Fig. We the with and of the free bile acids where from the reaction by soluble We from to by and catalyzed reaction to the to the also or was in the (Fig. This suggests that the baiF gene product CoA from to We then to the substrate for the baiF and baiK gene products secondary bile acid CoA and primary bile acid The bile acids CA and were of secondary bile acid CoA 1). and CA had reaction was however, to was was This was with both the baiF and baiK gene both the baiF and the baiK gene products were to CoA to the baiK had the reaction with this bile the gene product CoA transferase with and not with of were were with of from to and with product not We the bile acid which had a to for the baiF gene product, and it was to a by the baiK gene In addition, the baiF gene product, the baiK gene product to was was the was 1). We to a broad substrate as CDCA, are in human bile and the secondary bile acids and the of are by human bile acid bacteria. In the current study, we cloned and expressed two genes encoding bile acid CoA transferases from C. scindens VPI 12708 in E. We of secondary bile acid CoA and primary bile acid CoA and found that the baiF and baiK gene products have broad bile acid substrate specificity. However, the baiK gene product had was as the CoA 1). the bile acid CoA transferase a secondary bile acid was as the bile acid CoA cholic acid was the was as the CoA Bile acid 7α-dehydroxylating bacteria a of genes in the of primary bile acids (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). Upon the bile acids are rapidly conjugated to Previously, we showed that the baiB gene product ligated CoA to primary bile acids by an ATP-dependent manner (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). We also that the baiF gene product the of (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, in we not for CoA transferase activity. In proposed a new family of CoA transferases based on amino acid sequence and of reaction the not be into family or J. A new family of CoA-transferases.FEBS Lett. 2001; 509: 345-349Crossref PubMed Scopus (109) Google Scholar). This involved in anaerobic the baiF gene and has been to as the anaerobic is for to by ATP-dependent ligation by the ATP-dependent CoA ligation is to be to a pool of that be by an ATP-independent encoded by the gene product J. A new family of CoA-transferases.FEBS Lett. 2001; 509: 345-349Crossref PubMed Scopus (109) Google Scholar). In bile acid the baiB gene product to a pool of bile which then be on by the bile acid CoA transferases encoded by the baiF and baiK genes (Fig. 1). Type III CoA transferases, as the and gene products from E. coli D. M.A. and mechanism of the of 1999; PubMed Scopus Google Scholar, S. C. substrate and of Escherichia coli and A Bacteriol. 2008; PubMed Scopus Google Scholar, of Escherichia coli and with CoA and 2005; PubMed Scopus Google Scholar) as well as the transferase from S. S. and characterization of the transferase from Full Text Full Text PDF PubMed Scopus Google Scholar), a involved in of as well as CoA The baiF and baiK gene products this (Fig. has been proposed that CoA through S. S. and characterization of the transferase from Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. transferase the CoA the of an J. PubMed Scopus Google Scholar, J. of Escherichia coli a type III CoA PubMed Scopus Google Scholar, C. C. The of the E. coli gene product a new of two a family of CoA Full Text Full Text PDF PubMed Scopus Google Scholar). of in the from in of or decrease in specific S. S. and characterization of the transferase from Full Text Full Text PDF PubMed Scopus Google Scholar). a mechanism has been proposed based on for from of the mechanism of a III 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). The proposed mechanism for suggests that an acid with in of and a glycine and and from the The second then the the second which is in a by and This mechanism suggests that a is not formed as in family the reaction is before reaction products are of the mechanism of a III 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). of with CoA in the of the of Escherichia coli and with CoA and 2005; PubMed Scopus Google Scholar). This a for CoA shown for the baiF gene product in the of CoA substrate (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). We that a mechanism is involved in bile acid CoA by the baiF and the baiK genes to that proposed for the However, the baiF and baiK gene products to the glycine they use a to from We have that the baiF and baiK gene products have bile acid CoA transferase with broad bile acid substrate specificity. However, in the current study, we on secondary bile acid is that including and of and are the of and of the baiF and baiK genes be to this of genes in the of the pathway and of free bile acid bile of the step in the pathway where CoA is Previously, we isolated a of the from cell of of C. scindens VPI 12708 White W.B. J. Hylemon P.B. of a bile acid and mechanism of by an intestinal Eubacterium Full Text PDF PubMed Google Scholar). This suggests that CoA is in the however, be to which intermediate is the donor. We have discovered a operon that appears to be involved in bile acid metabolism in C. scindens VPI 12708 and C. hylemonae DSM 15053. The gene is to and and the is to encode a 3α-hydroxysteroid The baiK gene on this operon encodes a bile acid CoA transferase that may be to enhance transferase bile acids are of the baiK gene with genes in the of the pathway may a ATP-independent of CoA to of in this anaerobic genes this operon have been identified in the 7α-dehydroxylating C. hylemonae DSM C. scindens VPI and C. DSM genes were not in the 7α-dehydroxylating strain C. scindens ATCC be to the function of this In we report the first bile acid CoA transferases found in bacteria and suggest they function to conserve an ATP in the bile acid pathway (Fig. 1). The and for acid acid bile acid inducible cholic acid chenodeoxycholic acid deoxycholic acid transferase dehydrogenase lithocholic acid acid