Design of artificial nucleases is essential in biotechnology and biomedicine, whereas few artificial nucleases can both cleave and degrade DNA molecules. Heme proteins are potential enzymes for DNA cleavage. Using a small heme protein, myoglobin (Mb), as a model protein, we engineered a metal-binding motif of [1-His-1-Glu] (native His64 and mutated Glu29) in the heme distal site. The single mutant of L29E Mb was capable of not only efficient DNA cleavage but also DNA degradation upon Mg²⁺ binding to the heme distal site, as shown by an X-ray crystal structure of the Mg²⁺-L29E Mb complex. Molecular docking of the protein–DNA complex revealed multiple hydrogen-bonding interactions at their interfaces, involving both minor and major grooves of DNA. Moreover, both the distal Arg45 and the ligand Glu29 were identified as critical residues for the nuclease activity. This study reports the structure of a water-bridged heterodinuclear center of Mg-heme (Mg²⁺-H₂O-Fe³⁺), showing a similar function as the homodinuclear center (Mg_A²⁺-H₂O–Mg_B²⁺) in natural nuclease, which indicates that the Mg²⁺-L29E Mb complex is an effective artificial nuclease.

中文翻译：

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通过工程化肌红蛋白中镁血红素的异双核中心合理设计人工核酸酶。

人工核酸酶的设计在生物技术和生物医学中必不可少，而很少有人工核酸酶能够裂解和降解DNA分子。血红素蛋白是用于DNA切割的潜在酶。使用小的血红素蛋白，肌红蛋白（Mb）作为模型蛋白，我们在血红素远端位点设计了[1-His-1-Glu]（天然His64和突变的Glu29）的金属结合基序。L29E MB的单突变体不仅能够有效的DNA切割而且还取决于镁DNA降解²⁺结合血红素远端部位，如图中的镁的X射线晶体结构²⁺-L29E Mb复合物。蛋白质-DNA复合物的分子对接揭示了它们界面上的多个氢键相互作用，涉及DNA的主要和次要凹槽。此外，远端Arg45和配体Glu29都被确定为核酸酶活性的关键残基。这项研究报告了镁血红素的水桥异双核中心（Mg ²⁺ -H ₂ O-Fe ³⁺）的结构，其功能与同核双中心（Mg _A ²⁺ -H ₂ O-Mg _B ²⁺）在天然核酸酶中，这表明Mg ²⁺ -L29E Mb复合物是有效的人工核酸酶。