The biochemical characterization of a TatD nuclease from Thermus thermophilus

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2024-07-14 DOI:10.1016/j.pep.2024.106557

Yi-Xuan Zhao , Xiao Xiang , Xi-Peng Liu

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Abstract

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 Mg²⁺ and Mn²⁺. 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.

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嗜热菌 TatD 核酸酶的生化特征。

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领域也有多种应用。在核酸酶中，TatD 是一种外切核酸酶，在不同生物体内具有多种生物学功能。在此，我们对嗜热菌中潜在的 TatD 核酸酶进行了生物化学鉴定。我们克隆了嗜热菌的 tatD 基因，然后表达并纯化了重组的 TatD 核酸酶。我们的研究结果表明，TthTatD核酸酶既能降解单链DNA，也能降解双链DNA，而且其活性依赖于二价金属离子Mg2+和Mn2+。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 来自嗜热细菌，但它并不是一种恒温酶。根据 DNA 底物与 TthTatD 结构模型的序列相似性和分子对接，确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证实了它们的作用。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应用提供了主要依据。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.