BaiJ and BaiB are key enzymes in the chenodeoxycholic acid 7α-dehydroxylation pathway in the gut microbe Clostridium scindens ATCC 35704

Figures & data

Figure 1. Bile acid 7-dehydroxylation by C. scindens ATCC 35704.(a) CA 7α-dehydroxylation proposed by Funabashi and colleagues⁹ (the six-enzyme pathway) projected onto CDCA. (b) bai operon along with other genes known or suspected to be involved in bile acid transformations in C. scindens ATCC 35704 and the function of the encoded proteins. All enzymes were purified except BaiG.

Figure 2. 7α-dehydroxylation of CDCA using different enzyme combinations in vitro.

(a) Bile acids detected after incubation of six-enzyme set (BaiB, BaiCD, BaiE, BaiA2, BaiF, BaiH) or negative control (NEC, no-enzyme control) with 100 μM CDCA at different times. Only CDCA was detected for NEC. Minor amounts of LCA (0.24 ± 0.01 μM) was detected at 24 hours. 3-oxoLCA was detected starting at 1 hour with a maximum at 24 hours (5.68 ± 0.86 μM). No 3-oxoΔ⁴-CDCA, 3-oxo-Δ^4.6-LCA, or 3-oxo-Δ⁴-LCA was detected. (b) Bile acids detected after 22-24 hours incubation of 100 μM CDCA with the base enzyme set amended with additional oxidoreductase(s), as indicated below the bars. No 3-oxo-Δ⁴-CDCA or allo-LCA were detected in any sample. A minor amount of 3-oxo-Δ⁴-LCA (0.26 ± 0.44 μM) was detected only with one combination of enzymes (BaiB, CD, E, A2, H, J). In addition to 3-oxoLCA (14.51 ± 7.82 μM to 21.44 ± 0.39 μM), minor amounts of 3-oxo-allo-LCA (0.37 ± 0.32 μM to 1.38 ± 0.15 μM) and LCA (0.08 ± 0.02 μM to 0.47 ± 0.03 μM) were detected in all enzyme mixes containing BaiJ. Histograms depict the mean and standard deviation of three assays. Incomplete mass balance is attributed to the formation of CoA conjugates that are not quantifiable due to the absence of standards. The structure of all bile acids discussed here is listed in Table S1. Allo-bile acids were not quantified for data in panel A.

Figure 3. Conversion of CDCA over time with five-enzyme set (BaiB, BaiCD, BaiE, BaiA2, BaiJ).

Bile acids detected after incubation of five-enzyme set with 100 μM CDCA and sampled at various times. Only CDCA was detected for no-enzyme control (NEC, 24 hours). LCA and 3-oxoLCA were detected starting at 2 hours. No 3-oxo-Δ⁴-CDCA, 3-oxo-Δ^4,6-LCA, 3-oxo-Δ⁴-LCA or allo-bile acids were detected. Histograms depict the mean and standard deviation of three assays. Incomplete mass balances (between 10 minutes and 9 hours) are attributed to the formation of CoA conjugates that are not quantifiable due to the absence of standards. CoA conjugated bile acids are shown in Figure S2. The structure of all bile acids discussed here is listed in Table S1.

Figure 4. Conversion of CDCA by E. coli expressing bai genes.

Concentration of selected bile acids detected after incubation of engineered E. coli strains with 200 μM CDCA and sampled after 24 hours. Only detected intermediates of the 7α-dehydroxylation pathway are shown to allow visualization of bile acids found in low amounts. Concentrations of all detected bile acids are shown in Figure S4B. The bai genes in each strain are shown below bars. The last bar corresponds to the five-enzyme set plus BaiG. Histograms depict the mean and standard deviation of three assays. The structure of all bile acids discussed here is listed in Table S1.

Figure 5. Conversion of CDCA during sequential addition of five-enzyme set enzymes.

(a) Bile acid concentrations after incubation of 100 μM CDCA with indicated enzyme(s) for 90 min (150 min for BaiB) after which the next enzyme was added. Only bile acids in the proposed pathway are shown, but 7-oxoLCA and 3,7-dioxo-CDCA were also detected. Only CDCA was detected for no-enzyme control (not shown). No 3-oxo-Δ⁴-CDCA, 3-oxo-Δ^4,6-LCA, 3-oxo-Δ⁴-LCA or allo-bile acids were detected. Histograms depict the mean and standard deviation of three assays. The structure of the bile acids discussed here is listed in Table S1. (b) CoA conjugated bile acids detected at each step. CoA intermediates with * are only identified by their mass, whereas the others were identified on the basis on enzymatically produced standards. 3-oxoLCA-CoA could not be detected due a technical problem.

Figure 6. First reductive step in 7α-dehydroxylation pathway.

Bile acid concentrations after incubation of 3-oxo-Δ^4,6-LCA with BaiJ from strain ATCC 35704 for 3 hours. A minor amount of 3-oxo-Δ⁴-CDCA was seen in both samples (2.41 ± 0.07 μM and 2.21 ± 0.08 μM), indicating a slight enzyme-independent transformation (not shown in graph). Allo-bile acids were not assessed as they are not relevant for the first reductive step but a minor amount of a bile acid observed with BaiJ (not shown in graph and assigned as 3-oxo-LCA in data file), is believed to be 3-oxo-allo-LCA. Histograms depict the mean and standard deviation of three assays. Structures of bile acids are shown on the right.

Figure 7. Second reductive step in 7α-dehydroxylation pathway.

(a) 3-oxo-Δ⁴-LCA was incubated with BaiCD for 24 hours and bile acids quantified. Only a small amount of 3-oxoLCA (0.40 ± 0.04 μM) was detected with BaiCD. A slight contamination with 3,7-dioxoCDCA was observed in both assays (not shown). (b) 3-oxo-Δ⁴-LCA was incubated with BaiJ for 3 hours and bile acids quantified. Only 3-oxo-Δ^4,6-LCA, 3-oxo-Δ⁴-LCA and 3-oxo-allo-LCA were detected. Histograms depict the mean and standard deviation of three assays. Structures of bile acids are shown on the right.

Figure 8. Second reductive step with CoA-ligated bile acids.

Part A: 3-oxo-Δ⁴-CDCA was incubated for 18 hours in buffer containing CoA and with or without BaiB and bile acids quantified (only 3-oxo-Δ⁴-CDCA was detected, apart from a minor amount of 3-oxo-Δ^4,6-LCA seen without BaiB and in the NEC, possibly due to enzyme-independent dehydration). Part B: Each incubation was amended with additional enzymes (BaiE+BaiJ or BaiE+BaiJ+BaiCD) and cofactors and bile acids were quantified after 7 hours. 3-oxoLCA (0.53 ± 0.02 μM) was only detected in the assay with BaiB+BaiE+BaiJ+BaiCD. Histograms depict the mean and standard deviation of three assays. The structure of bile acids discussed here is listed in Table S1.

Figure 9. BaiJ from C. scindens strain VPI 12,708 cannot initiate reductive part of 7α-dehydroxylation pathway.

(a) Bile acid concentrations after incubation of 100 μM 3-oxo-Δ^4,6-LCA or 3-oxo-Δ⁴-LCA with BaiJ from C. scindens VPI 12708 for 3 hours. Structures of bile acids for the reactions are shown below graph. A minor amount of 3-oxo-Δ⁴-CDCA (2.21 ± 0.08 μM) was observed in the NEC with 3-oxo-Δ^4,6-LCA as substrate (not shown in graph). (b) Five-enzyme set with BaiJ from either strain ATCC 35704 or VPI 12708 (as indicated) incubated for 24 hours with 100 μM CDCA. No 3-oxo-allo-LCA, 3-oxoLCA or LCA was produced with BaiJ from strain VPI 12708. Only CDCA was detected in the NEC (not shown). Histograms depict the mean and standard deviation of three assays.

Figure 10. Third reductive step in 7α-dehydroxylation pathway.

Bile acid concentrations after incubation of (a) 3-oxoLCA for 3 hours or (b) 3-oxo-allo-LCA for 1 hour with indicated enzymes. Only 3-oxo-allo-LCA was detected in the NEC (panel B, not shown) after 24 hours incubation. Histograms depict the mean and standard deviation of three assays. Structures of bile acids are shown on the right.

Figure 11. Model of CDCA 7α-dehydroxylation in C. scindens ATCC 35704.

CDCA is transformed to 3-oxo-Δ⁴-CDCA-CoA by BaiB, BaiA2, and BaiCD, followed by dehydration catalyzed by BaiE to form 3-oxo-Δ^4,6-LCA-CoA, as described previously.⁹ The next steps in the published 7α-dehydroxylation pathway based on bai operon-encoded six-enzyme set are shown on the left.⁹ In our model of CDCA 7α-dehydroxylation with the C. scindens ATCC 35704 five-enzyme set (middle panel), the first reductive step directly follows the dehydration step and is catalyzed by BaiJ (and is reversible). The second reductive step is most likely catalyzed by BaiCD (as BaiCD is necessary to form 3-oxoLCA when starting from 3-oxo-Δ⁴-CDCA, Figure 8). The final reductive step can be catalyzed by either BaiA2 or BaiA1/3. CoA is expected to be lost only after the third reductive step (as we observed the presence of LCA-CoA), resulting in production of LCA. The final product LCA may be re-oxidized to 3-oxo-LCA by either BaiA2 or BaiA1/3. Without BaiB (right panel), a minor fraction of bile acids may proceed through the pathway and unconjugated 3-oxo-Δ^4,6-LCA enters the reductive branch where BaiJ catalyzes both the first and second reductive step resulting in the formation of 3-oxo-allo-LCA, with the possibility of further reduction to allo-LCA by either BaiA2 or BaiA1/3.

Funabashi M, Grove TL, Wang M, Varma Y, McFadden ME, Brown LC, Guo C, Higginbottom S, Almo SC, Fischbach MA. A metabolic pathway for bile acid dehydroxylation by the gut microbiome. Nature. 2020;582(7813):566–570. doi:10.1038/s41586-020-2396-4.

 PubMed Web of Science ®Google Scholar

Data availability statement

The data used in this manuscript are available at DOI 10.5281/zenodo.8263047