The biochemical characterization of a TatD nuclease from Thermus thermophilus.

Autor: Zhao YX; SJTU Yazhou Bay Institute of Deepsea Sci-Tech, Yongyou Industrial Park, Sanya, 572024, China., Xiang X; 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. Electronic address: zjxiao2018@sjtu.edu.cn., Liu XP; 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. Electronic address: xpliu@sjtu.edu.cn.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2024 Nov; Vol. 223, pp. 106557. Date of Electronic Publication: 2024 Jul 14.
DOI: 10.1016/j.pep.2024.106557
Abstrakt: 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 2+ and Mn 2+ . 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.
Competing Interests: Declaration of competing interest The authors declare that no conflicts of interest exist.
(Copyright © 2024. Published by Elsevier Inc.)
Databáze: MEDLINE
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