Biochemical characterization and mutational studies of a novel 3-meth-lyadenine DNA glycosylase II from the hyperthermophilic Thermococcus gammatolerans

The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes a putative 3-methlyadenine DNA glycosylase II (Tg-AlkA). Herein, we report biochemical characterization and catalytic mechanism of Tg-AlkA. The recombinant Tg-AlkA can excise hypoxanthine (Hx) and 1-methlyadenine (1-meA) from dsDNA with varied efficiencies at high temperature. Notably, Tg-AlkA is a bi-functional glycosylase, which is sharply distinct from all the reported AlkAs. Biochemical data show that the optimal temperature and pH of Tg-AlkA for removing Hx from dsDNA are ca.70 degrees C and ca.7.0-8.0, respectively. Furthermore, the Tg-AlkA activity is independent of a divalent metal ion, and Mg2+ stimulates the Tg-AlkA activity whereas other divalent ions inhibit the enzyme activity with varied degrees. Mutational studies show that the Tg-AlkA W204A and D223A mutants abolish completely the excision activity, thereby suggesting that residues W204 and D223 are involved in catalysis. Surprisingly, the mutations of W204, D223, Y139 and W256 to alanine in Tg-AlkA lead to the increased affinity for binding DNA substrate with varied degrees, suggesting that these residues are flexible for conformational change of the enzyme. Therefore, Tg-AlkA is a novel AlkA that can remove Hx and 1-meA from dsDNA, thus providing insights into repair of deaminated and alkylated bases in DNA from hyperthermophilic Thermococcus.

Automated summary

This study reports the biochemical characterization and catalytic mechanism of Tg-AlkA from the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans. Tg-AlkA is a bi-functional glycosylase with distinct features, and mutational studies reveal crucial catalytic residues and conformational flexibility.