Department of Biochemistry, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Japan
Bioorganic Research Institute, Suntory Foundation for Life Sciences, Kyoto, Japan.
Life Sci Alliance. 2023 Jul 24;6(10). doi: 10.26508/lsa.202302001. Print 2023 Oct.
DNA mismatch repair endonuclease MutL binds two zinc ions. However, the endonuclease activity of MutL is drastically enhanced by other divalent metals such as manganese, implying that MutL binds another catalytic metal at some site other than the zinc-binding sites. Here, we solved the crystal structure of the endonuclease domain of MutL in the manganese- or cadmium-bound form, revealing that these metals compete with zinc at the same sites. Mass spectrometry revealed that the MutL yielded 5'-phosphate and 3'-OH products, which is characteristic of the two-metal-ion mechanism. Crystallographic analyses also showed that the position and flexibility of a highly conserved Arg of MutL altered depending on the presence of zinc/manganese or the specific inhibitor cadmium. Site-directed mutagenesis revealed that the Arg was critical for the catalysis. We propose that zinc ion and its binding sites are physiologically of catalytic importance and that the two-metal-ion mechanism works in the reaction, where the Arg plays a catalytic role. Our results also provide a mechanistic insight into the inhibitory effect of a mutagen/carcinogen, cadmium, on MutL.
DNA 错配修复内切酶 MutL 结合两个锌离子。然而，MutL 的内切酶活性被其他二价金属如锰大大增强，这表明 MutL 在锌结合位点以外的某些部位结合另一个催化金属。在这里，我们解决了 MutL 的内切酶结构域在锰或镉结合形式下的晶体结构，揭示了这些金属与锌在相同的部位竞争。质谱分析显示 MutL 产生 5'-磷酸和 3'-OH 产物，这是双金属离子机制的特征。晶体结构分析还表明，MutL 的一个高度保守的精氨酸的位置和灵活性取决于锌/锰的存在或特定抑制剂镉的存在。定点突变显示精氨酸对于催化至关重要。我们提出锌离子及其结合位点在生理上具有催化重要性，并且双金属离子机制在反应中起作用，其中精氨酸发挥催化作用。我们的结果还为诱变剂/致癌剂镉对 MutL 的抑制作用提供了机制上的见解。
