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J. Biol. Chem., Vol. 282, Issue 50, 36514-36524, December 14, 2007

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Identification of Aspartic Acid and Histidine Residues Mediating the Reaction Mechanism and the Substrate Specificity of the Human UDP-glucuronosyltransferases 1A^*

Dong Li, Sylvie Fournel-Gigleux, Lydia Barré, Guillermo Mulliert, Patrick Netter, Jacques Magdalou, and Mohamed Ouzzine¹

From the UMR 7561, Faculté de Médecine and UMR 7036, Faculté des Sciences, CNRS-Université Henri Poincaré Nancy 1, 54505 Vandoeuvre-lès-Nancy, France

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.

Received for publication, April 12, 2007 , and in revised form, October 11, 2007.

^* This work was supported by grants from the Agence Nationale de la Recherche and the Ligue Régionale contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: UMR CNRS 7561-Université Henri Poincaré Nancy 1, Faculté de Médecine, BP 184, 54505 Vandœuvrelès-Nancy, France. Tel.: 33-3-83-68-39-72; Fax: 33-3-83-68-39-59; E-mail: ouzzine{at}medecine.uhp-nancy.fr.

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