Identification of aspartic acid and histidine residues mediating the reaction mechanism and the substrate specificity of the human UDP-glucuronosyltransferases 1A
Identification of aspartic acid and histidine residues mediating the reaction mechanism and the substrate specificity of the human UDP-glucuronosyltransferases 1A
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DOI：
10.1074/jbc.m703107200
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发表时间：
2007-12-14
影响因子：
4.8
通讯作者：
Ouzzine, Mohamed
中科院分区：
文献类型：
--
作者：
Li, Dong;Fournel-Gigleux, Sylvie;Ouzzine, Mohamed
人 UDP-葡萄糖醛酸基转移酶 UGT1A6 是主要的酚代谢 UDP-葡萄糖醛酸基转移酶亚型。它催化酚类异生物质对 UDP-葡萄糖醛酸的亲核攻击，导致水溶性葡萄糖醛酸的形成。该反应提出的催化机制是涉及天冬氨酸/谷氨酸和/或组氨酸残基的酸碱机制。在这里，我们通过定点诱变研究了 14 个高度保守的天冬氨酸/谷氨酸残基在人 UGT1A6 整个序列上的作用。我们表明，除了天冬氨酸残基 Asp-150 和 Asp-488 之外，丙氨酸取代羧基残基会产生活性突变体，但酶活性降低，对受体和/或供体底物的亲和力降低。包括其他 UGT1A 同工型中相应残基突变在内的进一步分析表明，Asp-150 发挥着主要的催化作用。在本报告中，我们还鉴定了一个对于 UGT1A 酶家族对酚类和羧酸底物的葡萄糖醛酸化非常重要的单个活性位点残基。用组氨酸取代 UGT1A4 的 Pro-40 扩大了酶对酚类和羧基化合物的葡萄糖醛酸化活性，因此，在底物特异性方面产生了 UGT1A3 型同工型。相反，当 UGT1A3 的 His-40 残基被脯氨酸取代时，底物特异性转向 UGT1A4，酚底物的葡萄糖醛酸化作用消失。此外，UGT1A1 的 His-39 残基（UGT1A4 中的 His-40）突变为脯氨酸会导致酚类葡萄糖醛酸化的丧失，但不会导致雌激素的葡萄糖醛酸化的丧失。这项研究为更好地理解葡萄糖醛酸化机制和底物识别迈出了一步，这对于更好地预测人体药物代谢和毒性具有不可估量的价值。
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.