Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
Novosibirsk State University, Novosibirsk, 630090, Russia.
Biochemistry (Mosc). 2020 Feb;85(2):192-204. doi: 10.1134/S0006297920020066.
8-Oxoguanine-DNA N-glycosylase (OGG1) is a eukaryotic DNA repair enzyme responsible for the removal of 8-oxoguanine (oxoG), one of the most abundant oxidative DNA lesions. OGG1 catalyzes two successive reactions - N-glycosidic bond hydrolysis (glycosylase activity) and DNA strand cleavage on the 3'-side of the lesion by β-elimination (lyase activity). The enzyme also exhibits lyase activity with substrates containing apurinic/apyrimidinic (AP) sites (deoxyribose moieties lacking the nucleobase). OGG1 is highly specific for the base opposite the lesion, efficiently excising oxoG and cleaving AP sites located opposite to C, but not opposite to A. The activity is also profoundly decreased by amino acid changes that sterically interfere with oxoG binding in the active site of the enzyme after the lesion is everted from the DNA duplex. Earlier, the molecular dynamics approach was used to study the conformational dynamics of such human OGG1 mutants in complexes with the oxoG:C-containing substrate DNA, and the population density of certain conformers of two OGG1 catalytic residues, Lys249 and Asp268, was suggested to determine the enzyme activity. Here, we report the study of molecular dynamics of human OGG1 bound to the oxoG:A-containing DNA and OGG1 mutants bound to the AP:C-containing DNA. We showed that the enzyme low activity is associated with a decrease in the populations of Lys249 and Asp268 properly configured for catalysis. The experimentally measured rate constants for the OGG1 mutants show a good agreement with the models. We conclude that the enzymatic activity of OGG1 is determined majorly by the population density of the catalytically competent conformations of the active site residues Lys249 and Asp268.
8-氧鸟嘌呤-DNA-N-糖基化酶（OGG1）是一种真核生物 DNA 修复酶，负责清除 8-氧鸟嘌呤（oxoG），这是最丰富的氧化 DNA 损伤之一。OGG1 催化两个连续的反应 - N-糖苷键水解（糖苷酶活性）和通过 β-消除（裂合酶活性）在损伤的 3'-侧切割 DNA 链。该酶还具有带有无嘌呤/无嘧啶（AP）位点（缺少核碱基的脱氧核糖部分）的底物的裂合酶活性。OGG1 对碱基对损伤具有高度特异性，有效切除 oxoG 并切割位于 C 但不位于 A 对面的 AP 位点。氨基酸变化也会极大地降低活性，这些变化会在损伤从 DNA 双链体中翻转后在酶的活性位点中产生空间干扰，从而阻止 oxoG 结合。早些时候，分子动力学方法被用于研究与含有 oxoG:C 的底物 DNA 结合的此类人类 OGG1 突变体的构象动力学，并且两个 OGG1 催化残基 Lys249 和 Asp268 的某些构象的种群密度被认为决定了酶的活性。在这里，我们报告了与人 OGG1 结合的含 oxoG:A 的 DNA 和与人 OGG1 突变体结合的含 AP:C 的 DNA 的分子动力学研究。我们表明，酶的低活性与适当构象的 Lys249 和 Asp268 的种群密度降低有关，这些构象有利于催化。实验测量的 OGG1 突变体的速率常数与模型吻合得很好。我们得出结论，OGG1 的酶活性主要由活性位点残基 Lys249 和 Asp268 的催化能力构象的种群密度决定。
