title: The critical role of amino acid residue at position 117 of mouse UDP-glucuronosyltransfererase 1a6a and 1a6b in resveratrol glucuronidation.
abstract: Mouse UDP-glucuronosyltransferase 1a6 (Ugt1a6) contains two functional copies of 1a6a and 1a6b that share high sequence homology (98%). Only 10 amino acids located around the substrate recognition region are different out of 531 total residues. Although Ugt1a6 plays important roles in conjugating phenolic compounds, the functional characteristics of these isozymes are unclear. We performed functional analyses of mouse Ugt1a6a and Ugt1a6b using two isomeric polyphenols (trans- and cis-resveratrol). The cDNAs of mouse Ugt1a6a and Ugt1a6b were cloned and constructed as recombinant proteins using a yeast expression system, and kinetic parameters were evaluated. The wild-type Ugt1a6a and Ugt1a6b proteins catalysed trans- and cis-resveratrol 3-O-glucuronidation. Although the K(m) value for trans-resveratrol was significantly lower for Ugt1a6a compared with Ugt1a6b, the K(m) values for cis-resveratrol were comparable for the isozymes. Despite high sequence homology, significant kinetic differences were observed between the isozymes. To identify the critical residues for resveratrol glucuronidation, we constructed 10 variants of Ugt1a6a (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E). The I117L variant had Ugt1a6b-like enzymatic properties of K(m) in trans-resveratrol, and V(max) and K(si) in cis-form, suggesting that the residues located at position 117 of Ugt1a6a and Ugt1a6b play an important role in resveratrol glucuronidation.
authors: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors: Shinsuke
authors: S
authors: Uchihashi
authors: Miyu
authors: M
authors: Nishikawa
authors: Toshiyuki
authors: T
authors: Sakaki
authors: Shin-ichi
authors: S
authors: Ikushiro
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D055162:Biocatalysis; D020719:Glucuronides / Q000737:chemistry / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D000077185:Resveratrol; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology; D013267:Stilbenes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D020719:Glucuronides; D007527:Isoenzymes; D013267:Stilbenes; D014453:Glucuronosyltransferase; D000077185:Resveratrol