Structure and functional characterization of a bile acid 7α dehydratase BaiE in secondary bile acid synthesis

Abstract

Conversion of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is performed by a few species of intestinal bacteria in the genus Clostridium through a multistep biochemical pathway that removes a 7α-hydroxyl group. The rate-determining enzyme in this pathway is bile acid 7α-dehydratase (baiE). In this study, crystal structures of apo-BaiE and its putative product-bound [3-oxo-Δ(4,6) -lithocholyl-Coenzyme A (CoA)] complex are reported. BaiE is a trimer with a twisted α + β barrel fold with similarity to the Nuclear Transport Factor 2 (NTF2) superfamily. Tyr30, Asp35, and His83 form a catalytic triad that is conserved across this family. Site-directed mutagenesis of BaiE from Clostridium scindens VPI 12708 confirm that these residues are essential for catalysis and also the importance of other conserved residues, Tyr54 and Arg146, which are involved in substrate binding and affect catalytic turnover. Steady-state kinetic studies reveal that the BaiE homologs are able to turn over 3-oxo-Δ(4) -bile acid and CoA-conjugated 3-oxo-Δ(4) -bile acid substrates with comparable efficiency questioning the role of CoA-conjugation in the bile acid metabolism pathway.

Keywords: 7α-dehyroxylation; bile acid 7α-dehydratase; gut microbe mediated human metabolite; gut microbes; nuclear transport factor-2 superfamily; primary bile acid; secondary bile acid; secondary bile acid synthesis; structural genomics.

Figure 1. Proposed steps involved in synthesis of secondary bile acid in human gut anaerobe, Clostridium sp

The oxidative and reductive arms of the pathway are enclosed within salmon and blue colored boxes, respectively. Enzymes with crystal structures solved using the JCSG pipeline are denoted in blue.

Figure 2. Structure of BaiE, bile acid 7α-dehydratase

(A) Secondary structure match (SSM) superposition of BaiE crystal structures from different organisms. The main chain atoms are colored gold, red and blue respectively for C. hiranonis DSM13275, C. scindens ATCC35704 and C. hylemonae DSM15053. (B) Characteristic twisted α+β barrel fold observed in BaiE. The secondary structure elements are colored according to C^α atomic B-value from blue (lowest B-value: 20 Ų) to red (highest B-value: 65 Ų). The blue and red arrows respectively indicate the start and end of loop residues 48–63. (C) Trimer assembly interface as observed in the crystal packing of BaiE, C. hiranonis DSM13275. Bound PEG molecules in the active site are indicated as sticks. The three subunits of the trimer assembly are colored yellow, cyan and green. The six atom, (His)₃– (H₂O)₃, octahedral coordination of the Ni ion is depicted in the square panel. The view in the square panel was obtained by ~90° clockwise rotation of the view of the trimer assembly in the primary figure around an axis perpendicular to the 3-fold symmetry. The coordinate bonds are depicted as solid black lines. N, O, and Ni atoms are colored blue, red and brown, respectively. The carbon atoms of the protein chain are colored yellow, cyan and green, respectively. (D) SSM superposition of BaiE, C. hiranonis DSM13275 (gold), onto closest structural homologues as determined by DALI. The proteins used in the superposition are two scytalone dehydrogenases (blue and cyan), LinA (magenta), an α+β barrel fold containing protein of unknown function (PDB ID: 3ROB) and Δ^5,3-ketosteroid isomerase (green). Two views between the figures are related ~90° anticlockwise rotation.

Figure 3. Substrate binding site of BaiE, bile acid 7α-dehydratase

(A) Location of the active site of each protomer with respect to the trimer assembly. The protomers of the trimer are colored gold, blue and green. (B) Cavities detected in the monomer structure of BaiE. Inner surface of the cavities are colored gray and the outer surface are colored black. The substrate binding site is indicated by the black arrow. (C) Key interaction involving residues of the substrate binding site with PEG and water molecules in BaiE, C. hiranonis DSM13275. 2Fo-Fc electron density map colored cyan contoured at 1.0 sigma. Dashed black lines are hydrogen bond interactions with numbers being distances in Å between the interacting atoms. Carbon atoms of protein residues and PEG molecule are colored gold and green, respectively. O, N, P and S atoms are colored red, blue and orange, respectively.

Figure 4. Predicted enzyme:substrate interactions

(A) Probable productive binding mode of 3-oxo-Δ⁴-Chenodeoxycholate (3-oxo-Δ⁴-CDCA). Blue dashed lines and adjacent numbers are predicted interaction of His83-Nε2 atom with C7-OH and C6 atoms and Y30-OH group with C3-oxo atom of 3-oxo-Δ⁴-CDCA. The 6α-H closest to H83-Nε2 atom colored magenta and 6β-H away from H83-Nε2 atom colored brown. (B) Predicted stacking interaction involving the adenine group of the Coenzyme (CoA) moiety of 3-oxo-Δ⁴-Chenodeoxycholyl CoA (3-oxo-Δ⁴-CDC-CoA) with Y115. The key interaction of the bile acid moiety of the docked CoA-bile acid ester with the active site residues is similar to what is predicted in (A). Carbon atoms of protein residues and product molecule are colored gold and green, respectively. H, O, N, P and S atoms are colored gray, red, blue, orange and olive, respectively.

Figure 5. Binding of product, 3-oxo-Δ^4,6- Lithocholyl-CoA (3-oxo-Δ^4,6-LC-CoA, in BaiE, C. hiranonis DSM13275

A) The extended binding pocket and binding of 3-oxo-Δ^4,6-LC-CoA, in BaiE, C. hiranonis DSM13275. Main chain of protomers arising from different trimer assemblies is colored gold and green, respectively. The loop formed by residues 48–63 is bordered by black and blue ellipses in the gold and green monomers, respectively. The black arrow indicates the location of the C3-oxo atom. (B) Key inter-protomer interactions involved in generating the extended binding pocket. Numbers indicate distances in Å. (C) Unbiased mFo-DFc electron density map of 3-oxo-Δ^4,6-LC-CoA contoured at +1.8 sigma (0.085 e⁻/ų). For clarity, both orientations of the ligand are shown separately to illustrate how the partial product model fits the density. A model with the two orientations together at partial occupancy fits the density better than either orientation on its own. The pyrophosphate and AMP moiety of the product is not shown. The density fit for those regions is not as good (see text). Product 3-oxo-Δ^4,6-LC-CoA in panels A and B is depicted as spheres.

Figure 6. In vitro generation of 3-oxo-Δ^4,6- lithocholyl-Coenzyme A from 3-oxo-Δ⁴-Chenodeoxycholyl CoA(3-oxo-Δ⁴-CDC-CoA) using purified BaiE from C. hiranonis DSM13275 as monitored by ¹H-NMR

A reaction mixture of 100μM 3-oxo-Δ⁴-CDC-CoA (3-oxo-Δ^4,6-LC-CoA) and 0.001μM BaiE was prepared in 20 mM HEPES pH 7.4 and incubated at 300°K. Selected regions from one-dimensional ¹H-NMR spectra that were acquired at the indicated times after mixing and temperature equilibration (bottom four spectra) are displayed. Reference spectra of 3-oxo-Δ⁴-CDC-CoA and 3-oxo-Δ^4,6-LC-CoA acquired in the same buffer are shown at the top labeled A and B respectively. Some representative peaks associated with 3-oxo-Δ⁴-CDC-CoA and 3-oxo-Δ^4,6-LC-CoA are marked with dashed and dotted lines respectively. The intensity of peaks associated with the substrate decrease with a concomitant increase in product peaks during the reaction. The spectrum of the product matches perfectly the spectra of3-oxo-Δ^4,6-LC-CoA . Examination of the amide region of the spectra (right hand panel) shows the appearance of two new peaks during the reaction and is associated with removal of the 7α-hydroxyl group and the generation of methene groups.

Figure 7

Proposed mechanism of catalysis by BaiE highlighting the role of catalytically important residues in elimination of the 7α-hydroxy group through release of a water molecule.