University of Helsinki, Finland.
Drug Metab Dispos. 2010 Apr;38(4):687-96. doi: 10.1124/dmd.109.031229. Epub 2010 Jan 20.
The amino acid sequences of the human UDP-glucuronosyltransferases (UGTs) 1A9 and 1A10 are 93% identical, yet there are large differences in their activity and substrate selectivity. For example, the regioselectivity in propranolol glucuronidation, the regioselectivity in dobutamine glucuronidation, and the glucuronidation rate of alpha- and beta-estradiol differ greatly between UGT1A9 and UGT1A10. To identify the residue responsible for the activity differences, we divided the N-terminal half of the two UGTs into five comparable segments by inserting four unique restriction sites at identical positions in both genes and constructing chimeras in which segments of UGT1A9 were individually replaced by the corresponding segments from UGT1A10. Activity analyses of the resulting mutants, 910A [1A10((1-83))/1A9((84-285))], 910B [1A9((1-83))/1A10((84-147))/1A9((148-285))], 910C [1A9((1-147))/1A10((148-181))/1A9((182-285))], 910D [1A9((1-181))/1A10((182-235))/1A9((236-285))], and 910E [1A9((1-235))/1A10((236-285))] indicated that more than one residue is responsible for the differences between UGT1A9 and UGT1A10. We next prepared four double chimeras, in which two of the above UGT1A9 segments were replaced simultaneously by the corresponding UGT1A10 segments. However, none of the double chimeras glucuronidated either estradiol, propranolol, or dobutamine at rates that resembled those of UGT1A10. On the other hand, studying the kinetics of 1-naphthol glucuronidation yielded more focused results, indicating that residues within segment B (84-147) contribute directly to the K(m) value for this substrate. Further mutagenesis and activity assays suggested that Phe117 of UGT1A9 participates in 1-naphthol binding. In addition, it appears that residues within segment C of the N-terminal domain, mainly at positions 152 and 169, contribute to the higher glucuronidation rates of UGT1A10.
人 UDP-葡糖醛酸基转移酶（UGTs）1A9 和 1A10 的氨基酸序列有 93%的同源性，但它们的活性和底物选择性有很大差异。例如，普萘洛尔葡糖醛酸化的区域选择性、多巴酚丁胺葡糖醛酸化的区域选择性以及α-和β-雌二醇的葡糖醛酸化速率在 UGT1A9 和 UGT1A10 之间有很大差异。为了确定导致活性差异的残基，我们通过在两个基因中相同位置插入四个独特的限制位点，将两个 UGT 的 N 端半部分分为五个可比片段，并构建嵌合体，其中 UGT1A9 的片段分别由 UGT1A10 的相应片段替换。对所得突变体 910A[1A10((1-83))/1A9((84-285))]、910B[1A9((1-83))/1A10((84-147))/1A9((148-285))]、910C[1A9((1-147))/1A10((148-181))/1A9((182-285))]、910D[1A9((1-181))/1A10((182-235))/1A9((236-285))]和 910E[1A9((1-235))/1A10((236-285))]的活性分析表明，有一个以上的残基负责 UGT1A9 和 UGT1A10 之间的差异。我们接下来制备了四个双嵌合体，其中两个上述 UGT1A9 片段同时被相应的 UGT1A10 片段替换。然而，没有一个双嵌合体以类似于 UGT1A10 的速率将雌二醇、普萘洛尔或多巴酚丁胺葡糖醛酸化。另一方面，研究 1-萘酚葡糖醛酸化的动力学得到了更集中的结果，表明 B 段（84-147）内的残基直接参与该底物的 K(m)值。进一步的突变和活性测定表明，UGT1A9 的苯丙氨酸 117 参与 1-萘酚结合。此外，似乎 N 端结构域 C 段内的残基（主要在位置 152 和 169）有助于 UGT1A10 更高的葡糖醛酸化速率。
