Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyamacho, Toyonaka, Osaka, 5608531, Japan.
Department of Materials Engineering Science, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyamacho, Toyonaka, Osaka, 5608531, Japan.
DNA Repair (Amst). 2020 Jun;90:102859. doi: 10.1016/j.dnarep.2020.102859. Epub 2020 Apr 28.
Endonuclease III (EndoIII) is nearly ubiquitous in all three domains of life. EndoIII family proteins exhibit a bifunctional (glycosylase/lyase) activity on oxidative/saturated pyrimidine bases, such as thymine glycol. Previous studies on EndoIII homologs have reported the presence of important residues involved in substrate binding and catalytic activity. However, a biochemical clarification of the roles of these residues as well as details of their evolutionary conservation is still lacking. This is particularly true for archaeal orthologs. The current study demonstrated the roles of the evolutionarily conserved residues of euryarchaeon Thermococcus kodakarensis EndoIII (TkoEndoIII). We utilized amino acid sequence analysis and homology modeling to identify highly conserved regions with potential key residues in the EndoIII proteins. Using Ala-substituted TkoEndoIII mutant proteins, residues of interest were quantitatively examined via DNA binding, glycosylase/AP lyase/bifunctional activity, and DNA trapping assays. The obtained results allowed us to determine the roles, as well as the significance of these roles in Schiff base formation (Lys140 as a nucleophile and Asp158), Tg recognition (His160), substrate binding (Arg59, Leu101, Trp102, and Gly136), β-elimination activities (Ser57 and Asp62), and [4Fe-4S] cluster formation (Cys208 and Cys215). Interestingly, a critical role played by the highly conserved Lys105 (predicted as being away from the catalytic site) in substrate binding, accompanied by a significant indirect effect on catalytic activity, were detected. Our results suggest that these particular residues play conserved roles among EndoIII orthologs across the domains. In addition to identifying the critical role of the highly conserved Lys105, the study provides a comprehensive understanding of the functions attributable to the evolutionarily conserved residues found in the EndoIII family, from Escherichia coli to humans.
内切核酸酶 III（EndoIII）几乎存在于所有生命的三个领域中。EndoIII 家族蛋白在氧化/饱和嘧啶碱基（如胸腺嘧啶二醇）上表现出双功能（糖苷酶/裂合酶）活性。以前对 EndoIII 同源物的研究报告了参与底物结合和催化活性的重要残基的存在。然而，这些残基的生化阐明以及它们进化保守性的细节仍然缺乏。对于古菌同源物来说尤其如此。本研究证明了广古菌 Thermococcus kodakarensis EndoIII（TkoEndoIII）的进化保守残基的作用。我们利用氨基酸序列分析和同源建模来鉴定 EndoIII 蛋白中具有潜在关键残基的高度保守区域。使用氨基酸取代的 TkoEndoIII 突变蛋白，通过 DNA 结合、糖苷酶/AP 裂合酶/双功能活性和 DNA 捕获测定定量检查感兴趣的残基。获得的结果使我们能够确定这些残基的作用及其在席夫碱形成中的重要性（Lys140 作为亲核试剂和 Asp158）、Tg 识别（His160）、底物结合（Arg59、Leu101、Trp102 和 Gly136）、β-消除活性（Ser57 和 Asp62）和[4Fe-4S]簇形成（Cys208 和 Cys215）。有趣的是，检测到高度保守的 Lys105（预测远离催化位点）在底物结合中发挥关键作用，并对催化活性产生显著的间接影响。我们的结果表明，这些特定的残基在整个领域的 EndoIII 同源物中发挥保守作用。除了确定高度保守的 Lys105 的关键作用外，该研究还提供了对从大肠杆菌到人在内的 EndoIII 家族中发现的进化保守残基所具有的功能的全面理解。
