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, we report crystal structures of apo-BaiE and its putative product-bound (3-oxo-Δ4,6- lithocholyl-Coenzyme A (CoA)) complex. 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 confirmed that these residues are essential for catalysis and also confirmed the importance of other conserved residues, Tyr54 and Arg146, which are involved in substrate binding and affect catalytic turnover. Steady state kinetic studies revealed that the BaiE homologs are able to turn over 3-oxo-Δ4-bile acid and CoA-conjugated 3-oxo-Δ4-bile acid substrates with comparable efficiency questioning the role of CoA-conjugation in the bile acid metabolism pathway.

Consequences of bile salt biotransformations by intestinal bacteria

“…80,81 The structures along with functional characterization provide important insights into the mechanisms of key biochemical steps of this pathway. Structural information is essential for designing small molecule inhibitors of key enzymes in this pathway.…”

“…65 The recently solved crystal structure of this enzyme revealed a trimeric assembly for all 3 key homologs whereas the monomers exhibit the canonical twisted a+b barrel fold characteristic of the Nuclear Transport Factor 2 (NTF2) family. 81 The trimers appear to be bonded together by a divalent metal cation, probably Zn 2+ . The binding of bile acid substrate to BA 7a-DeOH is shown in Figure 5 Sitedirected mutagenesis studies established the role of 3 conserved residues in the active site namely, Tyr30, Asp35 and His83, as important for catalysis (Figs.…”

“…In all, the apostructures of baiE residues Asp106 and Arg146 in the substrate binding pocket interact via a salt bridge. 81 Severance of the salt bridge by replacing with residues Asn and Gln at position 106 and 146, respectively, abolished catalysis. Furthermore, steady-state kinetic characterization studies revealed that unlike baiA1 and baiA2 all baiE homologs are able to turnover 3-oxo-D 4 -bile acid and 3-oxo-D 4 -bile acid-CoA thioester substrates with comparable efficiency.…”

An unexplored pathway for degradation of cholate requires a 7α‐hydroxysteroid dehydratase and contributes to a broad metabolic repertoire for the utilization of bile salts in Novosphingobium sp. strain Chol11

“…N ov2c400 was automatically annotated as a gene coding for a hypothetical protein and shares 38% sequence identity with bile acid 7α‐dehydratase BaiE from Clostridium scindens (Bhowmik et al ., ) at the amino acid level (Fig. ).…”

The presence and severity of nonalcoholic steatohepatitis is associated with specific changes in circulating bile acids

“…An alternate possibility is that a decrease in intestinal dehydroxylation of primary BAs and reduced formation of secondary BAs results in enhanced primary BA synthesis. These functions are dependent on the intestinal microbiota and have been extensively characterized . Another conceivable reason for increased plasma primary conjugated BAs in NASH may be due to decreased enterohepatic circulation and/or a decrease in small intestinal bacteria capable of deconjugating primary BAs.…”