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| H64M/C120S Ngb神经红蛋白的核酸酶活性研究 |
| Study on the Nuclease Activity of Ngb (Neuroglobin) Variant H64M/C120S |
| 投稿时间：2025-01-11 修订日期：2025-02-06 |
| DOI： |
| 中文关键词: 人工金属酶 神经红蛋白 人工核酸酶 DNA断裂 |
| 英文关键词:Artificial metalloenzyme Neuroglobin Artificial nuclease DNA cleavage |
| 基金项目:衡阳市科技计划项目(202222035774),湖南省自然科学基金(2022JJ30485) |
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| 中文摘要: |
| 人工核酸酶的研究是生物技术领域的一个重要研究方向。但目前关于人工神经红蛋白的核酸酶活性的研究非常少。我们纯化了双突变体H64M/C120S神经红蛋白（H64M/C120S Ngb），并对其核酸酶活性进行了研究。琼脂糖凝胶电泳结果显示，H64M/C120S Ngb可以在没有外加氧化还原试剂的情况下，有效地断裂DNA。同时，H64M/C120S Ngb在还原剂二硫苏糖醇（DTT）存在时，也能有效地氧化断裂双链DNA。进一步的研究发现，Mg2+金属离子能够有效地促进该蛋白的核酸酶活性。机理实验显示，H64M/C120S Ngb在不存在氧化还原试剂条件下通过水解方式断裂DNA。在还原剂DTT存在时，H64M/C120S Ngb通过水解和氧化两种方式断裂DNA。这为今后以神经红蛋白为模板的人工核酸酶的设计和研究提供了借鉴。 |
| 英文摘要: |
| Artificial nuclease research is an important research direction in the field of biotechnology and biomedicine. However, there is currently limited research on the nuclease activity of artificial neuroglobin. We have purified the double mutant H64M/C120S neuroglobin and studied its nuclease activity. Results from agarose gel electrophoresis indicate that the pentacoordinate H64M/C120S neuroglobin can effectively cleave DNA without the addition of reducing agents. Furthermore, in the presence of the reducing agent dithiothreitol (DTT), H64M/C120S neuroglobin can also effectively oxidatively cleave double-stranded DNA. Further investigations revealed that the metal ion Mg2+ can significantly enhance the nuclease activity of this protein. The mechanistic experiments revealed that H64M/C120S Ngb cleaves DNA through hydrolysis in the absence of redox reagents. In the presence of the reductant DTT, H64M/C120S Ngb cleaves DNA through both hydrolysis and oxidation. |
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