Unno Masaki, Morikawa Masayuki, Sychrovský Vladimír, Koga Masataka, Minowa Nozomi, Komuro Saki, Shimizu Mami, Fukuta Mariko, Tsuyuguchi Fuuka, Mano Haruka, Ochi Yusuke, Nakashima Katsuyuki, Okamoto Yasuko, Saio Tomohide, Hattori Yoshikazu, Tanaka Yoshiyuki
Graduate School of Science and Engineering, Ibaraki University, Hitachi, Ibaraki 316-8511, Japan.
Research and Education Center for Atomic Sciences, Ibaraki University, Naka-Tokai, Ibaraki 319-1106, Japan.
Nucleic Acids Res. 2025 Jul 19;53(14). doi: 10.1093/nar/gkaf718.
The human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a bifunctional DNA repair enzyme that possesses both glycosylase and AP-lyase activity. Its AP-lyase reaction mechanism had been revealed by crystallographic capturing of the intermediate adduct. However, no intermediate within the glycosylase reaction was reported to date and the relevant reaction mechanism thus remained unresolved. In this work, we studied the glycosylase reaction of hOGG1 by time-resolved crystallography and spectroscopic/enzymological analyses. To trigger the glycosylase reaction within a crystal, we created a pH-responsive mutant of hOGG1 in which lysine 249 (K249) has been replaced by histidine (H), and designated hOGG1(K249H). Using hOGG1(K249H), a reactive intermediate state of the hOGG1(K249H)-DNA complex was captured in crystal upon pH activation. An unprecedented, ribose-ring-opened hemiaminal structure at the 8-oxoguanine (oxoG) site was found. Based on the structure of the reaction intermediate and QM/MM (quantum mechanics/molecular mechanics) calculations, a glycosylase reaction pathway of hOGG1(K249H) was identified where the aspartic acid 268 (D268) acts as a proton donor to O4' of oxoG. Moreover, enzymologically derived pKa (4.5) of a catalytic residue indicated that the observed pKa can be attributed to the carboxy group of D268. Thus, a reaction mechanism of the glycosylase reaction by hOGG1(K249H) has been proposed.
人类8-氧代鸟嘌呤DNA糖基化酶1（hOGG1）是一种具有糖基化酶和AP裂解酶活性的双功能DNA修复酶。其AP裂解酶反应机制已通过中间加合物的晶体学捕获得以揭示。然而，迄今为止尚未报道糖基化酶反应中的中间体，因此相关反应机制仍未得到解决。在这项工作中，我们通过时间分辨晶体学以及光谱/酶学分析研究了hOGG1的糖基化酶反应。为了在晶体中引发糖基化酶反应，我们构建了hOGG1的pH响应突变体，其中赖氨酸249（K249）被组氨酸（H）取代，命名为hOGG1（K249H）。利用hOGG1（K249H），在pH激活后，hOGG1（K249H）-DNA复合物的反应性中间状态在晶体中被捕获。在8-氧代鸟嘌呤（oxoG）位点发现了一种前所未有的核糖环开环半缩醛胺结构。基于反应中间体的结构和量子力学/分子力学（QM/MM）计算，确定了hOGG1（K249H）的糖基化酶反应途径，其中天冬氨酸268（D268）作为oxoG的O4'的质子供体。此外，催化残基的酶学推导pKa（4.5）表明，观察到的pKa可归因于D268的羧基。因此，已提出hOGG1（K249H）的糖基化酶反应机制。
