Department of Inorganic, Organic and Analytical Chemistry, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary.
College of Pharmacy, University of Babylon, Hillah 51001, Iraq.
Molecules. 2023 Jul 19;28(14):5511. doi: 10.3390/molecules28145511.
The nuclease domain of colicin E7 cleaves double-strand DNA non-specifically. Zn ion was shown to be coordinated by the purified NColE7 as its native metal ion. Here, we study the structural and catalytic aspects of the interaction with Ni, Cu and Cd non-endogenous metal ions and the consequences of their competition with Zn ions, using circular dichroism spectroscopy and intact protein mass spectrometry. An R447G mutant exerting decreased activity allowed for the detection of nuclease action against pUC119 plasmid DNA via agarose gel electrophoresis in the presence of comparable metal ion concentrations. It was shown that all of the added metal ions could bind to the apoprotein, resulting in a minor secondary structure change, but drastically shifting the charge distribution of the protein. Zn ions could not be replaced by Ni, Cu and Cd. The nuclease activity of the Ni-bound enzyme was extremely high in comparison with the other metal-bound forms, and could not be inhibited by the excess of Ni ions. At the same time, this activity was significantly decreased in the presence of equivalent Zn, independent of the order of addition of each component of the mixture. We concluded that the Ni ions promoted the DNA cleavage of the enzyme through a more efficient mechanism than the native Zn ions, as they directly generate the nucleophilic OH ion.
大肠杆菌素 E7 的核酸酶结构域能够非特异性地切割双链 DNA。研究表明，锌离子是天然存在于纯化的 NColE7 中的配位离子。在这里，我们使用圆二色性光谱法和完整蛋白质质谱法研究了与非内源性金属离子 Ni、Cu 和 Cd 的相互作用的结构和催化方面，以及它们与锌离子竞争的后果。一个活性降低的 R447G 突变体，在比较金属离子浓度的情况下，通过琼脂糖凝胶电泳检测到了对 pUC119 质粒 DNA 的核酸酶作用。结果表明，所有添加的金属离子都可以与脱辅基蛋白结合，导致较小的二级结构变化，但明显改变了蛋白质的电荷分布。锌离子不能被 Ni、Cu 和 Cd 取代。与其他金属结合形式相比，Ni 结合酶的核酸酶活性非常高，并且不能被过量的 Ni 离子抑制。同时，在存在等效 Zn 的情况下，这种活性显著降低，而与混合物中各成分的添加顺序无关。我们得出结论，Ni 离子通过比天然 Zn 离子更有效的机制促进了酶的 DNA 切割，因为它们直接生成亲核 OH 离子。
