Identification and characterization of two bile acid coenzyme A transferases from Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium

Abstract

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.

Fig. 1.

Current model of the bile acid 7α-dehydroxylation pathway in Clostridium scindens VPI 12708.

Fig. 2.

Schematic representation of bile acid 7α-dehydroxylation operons from C. scindens VPI 12708 and C. hylemonae DSM 15053. “P” represents putative promoter regions.

Fig. 3.

Schematic representation of baiJKL operon from Clostridium scindens VPI 12708 and Clostridium hylemonae DSM 15053. The baiA genes encode 27 kDa 3α-hydroxysteroid dehydrogenases. The baiJ genes encode a predicted 62 kDa flavoprotein similar to 3-ketosteroid-Δ¹-dehydrogenases. The baiK genes encode a predicted 49 kDa type III CoA transferase homologous to the baiF gene. The baiL genes are predicted to encode a 27 kDa protein in the short chain reductase family. “P” represents conserved bai “promoter” region. TspO/MBR family acts in signal transduction as well as in transport of steroids/dicarboxylic tetrapyrrole intermediates.

Fig. 4.

Boxshade 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.

Fig. 5.

Transcriptional analysis of baiJKL operons from C. hylemonae and C. scindens VPI 12708. A: Transcriptional initiation site for baiJ gene from C. hylemonae (underlined). B: Transcriptional initiation site for baiJ gene from C. scindens VPI 12708. C: Boxshade alignment of conserved putative regulatory elements upstream of the baiB between C. scindens VPI 12708, C. hylemonae DSM 15053, and C. hiranonis 13275 (above). The baiJ-conserved regulatory region from C. hylemonae DSM 15053 compared with that of the baiB upstream element (below) D: RT-PCR using primers spanning intergenic region of baiJ-baiK and baiK-baiL genes of C. scindens VPI 12708. “+” represents 10 ng C. scindens VPI 12708 genomic DNA, “−RT” represents negative control in which reverse transcriptase is not added, and “RT” represents cDNA prepared from uninduced (UI) and 50 μM cholic acid-induced (CA) cultures of C. scindens VPI 12708. M, methionine; rbs, ribosome binding site; TIS, transcription initiation site.

Fig. 6.

Purification of the streptavidin-tagged recombinant bile acid-CoA transferases from E. coli. A. Purification of the recombinant C-terminal streptavidin tagged BaiF. B. Purification of the recombinant C-terminal streptavidin tagged BaiK. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 30 μg crude (1) and 5g Streptactin-purified (2) fractions. Molecular weight standards are indicated to the left (M). (B) Detection of streptavidin-tagged recombinant transferases in crude (1) and purified (2) fractions using Strep-tag II antibody.

Fig. 7.

Reverse phase C-18 HPLC elution profile of CoA transferase reaction products catalyzed by BaiF. Reactions contained 500 μM cholic acid (17,600 DPM/nmole [24-14C] cholic acid) and 500 μM deoxycholyl-CoA A: No enzyme control elution profile. B: 0.01 μg BaiF catalyzed reaction. Reactions were acidified and extracted with ethyl acetate to remove free bile acids prior to HPLC. Radioactivity represented by triangles; absorption at 260 nm represented by dots.