SJTU Yazhou Bay Institute of Deepsea Sci-Tech, Yongyou Industrial Park, Sanya, 572024, China.
SJTU Yazhou Bay Institute of Deepsea Sci-Tech, Yongyou Industrial Park, Sanya, 572024, China; State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong-Chuan Road, Shanghai, 200240, China; Joint International Research Laboratory of Metabolic & Developmental Sciences (Ministry of Education), Shanghai Jiao Tong University, 800 Dong-Chuan Road, Shanghai, 200240, China; State Key Laboratory of Ocean Engineering, School of Naval Architecture, Ocean and Civil Engineering, Shanghai Jiao Tong University, 800 Dong-Chuan Road, Shanghai, 200240, China.
Protein Expr Purif. 2024 Nov;223:106557. doi: 10.1016/j.pep.2024.106557. Epub 2024 Jul 14.
Nucleases play pivotal roles in DNA repair and apoptosis. Moreover, they have various applications in biotechnology and industry. Among nucleases, TatD has been characterized as an exonuclease with various biological functions in different organisms. Here, we biochemically characterized the potential TatD nuclease from Thermus thermophilus. The tatD gene from T. thermophilus was cloned, then the recombinant TatD nuclease was expressed and purified. Our results revealed that the TthTatD nuclease could degrade both single-stranded and double-stranded DNA, and its activity is dependent on the divalent metal ions Mg and Mn. Remarkably, the activity of TthTatD nuclease is highest at 37 °C and decreases with increasing temperature. TthTatD is not a thermostable enzyme, even though it is from a thermophilic bacterium. Based on the sequence similarity and molecular docking of the DNA substrate into the modeled TthTatD structure, several key conserved residues were identified and their roles were confirmed by analyzing the enzymatic activities of the site-directed mutants. The residues E86 and H149 play key roles in binding metal ions, residues R124/K126 and K211/R212 had a critical role in binding DNA substrate. Our results confirm the enzymatic properties of TthTatD and provide a primary basis for its possible application in biotechnology.
核酸酶在 DNA 修复和细胞凋亡中发挥着关键作用。此外，它们在生物技术和工业中有多种应用。在核酸酶中，TatD 已被鉴定为一种具有不同生物功能的外切核酸酶，存在于不同的生物体中。在这里，我们对来自嗜热栖热菌的潜在 TatD 核酸酶进行了生化特性分析。我们从嗜热栖热菌中克隆了 tatD 基因，然后表达和纯化了重组 TatD 核酸酶。我们的结果表明，TthTatD 核酸酶可以降解单链和双链 DNA，其活性依赖于二价金属离子 Mg 和 Mn。值得注意的是，TthTatD 核酸酶的活性在 37°C 时最高，随着温度的升高而降低。尽管 TthTatD 来自嗜热菌，但它不是一种热稳定的酶。根据 DNA 底物与建模的 TthTatD 结构的序列相似性和分子对接，鉴定了几个关键的保守残基，并通过分析定点突变体的酶活性来证实了这些残基的作用。残基 E86 和 H149 在结合金属离子中起关键作用，残基 R124/K126 和 K211/R212 在结合 DNA 底物中起关键作用。我们的研究结果证实了 TthTatD 的酶学特性，并为其在生物技术中的可能应用提供了初步的基础。
