title: Identification of aspartic acid and histidine residues mediating the reaction mechanism and the substrate specificity of the human UDP-glucuronosyltransferases 1A.
abstract: The human UDP-glucuronosyltransferase UGT1A6 is the primary phenol-metabolizing UDP-glucuronosyltransferase isoform. It catalyzes the nucleophilic attack of phenolic xenobiotics on UDP-glucuronic acid, leading to the formation of water-soluble glucuronides. The catalytic mechanism proposed for this reaction is an acid-base mechanism that involves an aspartic/glutamic acid and/or histidine residue. Here, we investigated the role of 14 highly conserved aspartic/glutamic acid residues over the entire sequence of human UGT1A6 by site-directed mutagenesis. We showed that except for aspartic residues Asp-150 and Asp-488, the substitution of carboxylic residues by alanine led to active mutants but with decreased enzyme activity and lower affinity for acceptor and/or donor substrate. Further analysis including mutation of the corresponding residue in other UGT1A isoforms suggests that Asp-150 plays a major catalytic role. In this report we also identified a single active site residue important for glucuronidation of phenols and carboxylic acid substrates by UGT1A enzyme family. Replacing Pro-40 of UGT1A4 by histidine expanded the glucuronidation activity of the enzyme to phenolic and carboxylic compounds, therefore, leading to UGT1A3-type isoform in terms of substrate specificity. Conversely, when His-40 residue of UGT1A3 was replaced with proline, the substrate specificity shifted toward that of UGT1A4 with loss of glucuronidation of phenolic substrates. Furthermore, mutation of His-39 residue of UGT1A1 (His-40 in UGT1A4) to proline led to loss of glucuronidation of phenols but not of estrogens. This study provides a step forward to better understand the glucuronidation mechanism and substrate recognition, which is invaluable for a better prediction of drug metabolism and toxicity in human.
authors.affiliation: UMR 7561, Faculté de Médecine and UMR 7036, Faculté des Sciences, CNRS-Université Henri Poincaré Nancy 1, 54505 Vandoeuvre-lès-Nancy, France.
authors.forename: Dong
authors.initials: D
authors.lastname: Li
authors.forename: Sylvie
authors.initials: S
authors.lastname: Fournel-Gigleux
authors.forename: Lydia
authors.initials: L
authors.lastname: Barré
authors.forename: Guillermo
authors.initials: G
authors.lastname: Mulliert
authors.forename: Patrick
authors.initials: P
authors.lastname: Netter
authors.forename: Jacques
authors.initials: J
authors.lastname: Magdalou
authors.forename: Mohamed
authors.initials: M
authors.lastname: Ouzzine
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010636:Phenols / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D015262:Xenobiotics / Q000737:chemistry
chemical_list: D010636:Phenols; D015262:Xenobiotics; C019787:bilirubin glucuronoside glucuronosyltransferase; D001224:Aspartic Acid; D006639:Histidine; D020723:Glucuronic Acid; C110655:UDP-glucuronosyltransferase, UGT1A6; D014453:Glucuronosyltransferase