Waz Shaimaa, Nakamura Teruya, Hirata Keisuke, Koga-Ogawa Yukari, Chirifu Mami, Arimori Takao, Tamada Taro, Ikemizu Shinji, Nakabeppu Yusaku, Yamagata Yuriko
From the Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973.
the Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto 862-0973.
J Biol Chem. 2017 Feb 17;292(7):2785-2794. doi: 10.1074/jbc.M116.749713. Epub 2016 Dec 29.
The human MutT homolog 1 (hMTH1, human NUDT1) hydrolyzes oxidatively damaged nucleoside triphosphates and is the main enzyme responsible for nucleotide sanitization. hMTH1 recently has received attention as an anticancer target because hMTH1 blockade leads to accumulation of oxidized nucleotides in the cell, resulting in mutations and death of cancer cells. Unlike MutT, which shows high substrate specificity for 8-oxoguanine nucleotides, hMTH1 has broad substrate specificity for oxidized nucleotides, including 8-oxo-dGTP and 2-oxo-dATP. However, the reason for this broad substrate specificity remains unclear. Here, we determined crystal structures of hMTH1 in complex with 8-oxo-dGTP or 2-oxo-dATP at neutral pH. These structures based on high quality data showed that the base moieties of two substrates are located on the similar but not the same position in the substrate binding pocket and adopt a different hydrogen-bonding pattern, and both triphosphate moieties bind to the hMTH1 Nudix motif ( the hydrolase motif) similarly and align for the hydrolysis reaction. We also performed kinetic assays on the substrate-binding Asp-120 mutants (D120N and D120A), and determined their crystal structures in complex with the substrates. Analyses of bond lengths with high-resolution X-ray data and the relationship between the structure and enzymatic activity revealed that hMTH1 recognizes the different oxidized nucleotides via an exchange of the protonation state at two neighboring aspartate residues (Asp-119 and Asp-120) in its substrate binding pocket. To our knowledge, this mechanism of broad substrate recognition by enzymes has not been reported previously and may have relevance for anticancer drug development strategies targeting hMTH1.
人类MutT同源蛋白1（hMTH1，即人类NUDT1）可水解氧化损伤的核苷三磷酸，是负责核苷酸净化的主要酶。hMTH1最近作为一个抗癌靶点受到关注，因为阻断hMTH1会导致细胞内氧化核苷酸的积累，从而导致癌细胞发生突变并死亡。与对8 - 氧代鸟嘌呤核苷酸具有高底物特异性的MutT不同，hMTH1对包括8 - 氧代 - dGTP和2 - 氧代 - dATP在内的氧化核苷酸具有广泛的底物特异性。然而，这种广泛底物特异性的原因尚不清楚。在这里，我们确定了hMTH1在中性pH条件下与8 - 氧代 - dGTP或2 - 氧代 - dATP形成复合物的晶体结构。基于高质量数据的这些结构表明，两种底物的碱基部分位于底物结合口袋中相似但不完全相同的位置，并采用不同的氢键模式，且两个三磷酸部分与hMTH1的Nudix基序（水解酶基序）的结合方式相似，并为水解反应进行排列。我们还对底物结合位点的天冬氨酸120突变体（D120N和D120A）进行了动力学分析，并确定了它们与底物形成复合物的晶体结构。通过高分辨率X射线数据对键长的分析以及结构与酶活性之间的关系表明，hMTH1通过其底物结合口袋中两个相邻天冬氨酸残基（天冬氨酸119和天冬氨酸120）质子化状态的交换来识别不同的氧化核苷酸。据我们所知，酶的这种广泛底物识别机制以前尚未见报道，可能与靶向hMTH1的抗癌药物开发策略相关。
