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.

中文翻译：

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柯达热球菌核酸内切酶III的突变分析揭示了进化上保守的残基的作用。

核酸内切酶III（EndoIII）在生活的所有三个领域中几乎无处不在。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（糖基化酶/裂解酶）活性。以前对EndoIII同源物的研究已经报道了参与底物结合和催化活性的重要残基的存在。然而，仍缺乏对这些残基的作用及其进化保守性的细节的生化澄清。对于古细菌直系同源物尤其如此。当前的研究证明了在进化上保守的柯达氏嗜热球菌EndoIII（TkoEndoIII）残基的作用。我们利用氨基酸序列分析和同源性模型来鉴定EndoIII蛋白中具有潜在关键残基的高度保守区域。使用Ala取代的TkoEndoIII突变蛋白，通过DNA结合，糖基化酶/ AP裂解酶/双功能活性和DNA捕获测定法对目标残基进行了定量检查。获得的结果使我们能够确定席夫碱形成（Lys140作为亲核试剂和Asp158），Tg识别（His160），底物结合（Arg59，Leu101，Trp102和Gly136）的作用以及这些作用的意义， 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（预计远离催化位点）在底物结合中起着关键作用，并伴随着对催化活性的显着间接影响。我们的结果表明，这些特定残基在整个域的EndoIII直系同源基因中起保守作用。除了确定高度保守的Lys105的关键作用外，该研究还提供了对EndoIII家族（从大肠杆菌到人类）中进化上保守的残基的功能的全面理解。