Mutational analysis of Thermococcus kodakarensis Endonuclease III reveals the roles of evolutionarily conserved residues

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 Aspl 58), Tg recognition (His160), substrate binding (Arg59, Leu101, Trp102, and Gly136), beta-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.