title: Structural and functional characterization of BaiA, an enzyme involved in secondary bile acid synthesis in human gut microbe.
abstract: Despite significant influence of secondary bile acids on human health and disease, limited structural and biochemical information is available for the key gut microbial enzymes catalyzing its synthesis. Herein, we report apo- and cofactor bound crystal structures of BaiA2, a short chain dehydrogenase/reductase from Clostridium scindens VPI 12708 that represent the first protein structure of this pathway. The structures elucidated the basis of cofactor specificity and mechanism of proton relay. A conformational restriction involving Glu42 located in the cofactor binding site seems crucial in determining cofactor specificity. Limited flexibility of Glu42 results in imminent steric and electrostatic hindrance with 2'-phosphate group of NADP(H). Consistent with crystal structures, steady state kinetic characterization performed with both BaiA2 and BaiA1, a close homolog with 92% sequence identity, revealed specificity constant (kcat /KM ) of NADP(+) at least an order of magnitude lower than NAD(+) . Substitution of Glu42 with Ala improved specificity toward NADP(+) by 10-fold compared to wild type. The cofactor bound structure uncovered a novel nicotinamide-hydroxyl ion (NAD(+) -OH(-) ) adduct contraposing previously reported adducts. The OH(-) of the adduct in BaiA2 is distal to C4 atom of nicotinamide and proximal to 2'-hydroxyl group of the ribose moiety. Moreover, it is located at intermediary distances between terminal functional groups of active site residues Tyr157 (2.7 Å) and Lys161 (4.5 Å). Based on these observations, we propose an involvement of NAD(+) -OH(-) adduct in proton relay instead of hydride transfer as noted for previous adducts.
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, 92037; Joint Center for Structural Genomics.
authors.forename: Shiva
authors.initials: S
authors.lastname: Bhowmik
authors.forename: David H
authors.initials: DH
authors.lastname: Jones
authors.forename: Hsien-Po
authors.initials: HP
authors.lastname: Chiu
authors.forename: In-Hee
authors.initials: IH
authors.lastname: Park
authors.forename: Hsiu-Ju
authors.initials: HJ
authors.lastname: Chiu
authors.forename: Herbert L
authors.initials: HL
authors.lastname: Axelrod
authors.forename: Carol L
authors.initials: CL
authors.lastname: Farr
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Tien
authors.forename: Sanjay
authors.initials: S
authors.lastname: Agarwalla
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lesley
mesh_terms: D001051:Apoenzymes / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001647:Bile Acids and Salts / Q000096:biosynthesis*; D020134:Catalytic Domain; D003013:Clostridium / Q000201:enzymology*; D018360:Crystallography, X-Ray; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D009243:NAD / Q000737:chemistry
chemical_list: D001051:Apoenzymes; D001426:Bacterial Proteins; D001647:Bile Acids and Salts; D009243:NAD; D006913:Hydroxysteroid Dehydrogenases
keywords: gut microbe mediated human metabolite; nicotinamide-hydroxyl adduct; primary bile acid; proton relay; secondary bile acid; short-chain dehydrogenase/reductase; steroid dehydrogenase