Theoretical Insights into N-Glycoside Bond Cleavage of 5-Carboxycytosine by Thymine DNA Glycosylase: A QM/MM Study.

Thymine DNA glycosylase (TDG)-mediated excision of 5-formylcytosine and 5-carboxylcytosine (5-caC) is a critical step in active DNA demethylation. Herein, we employed a combined quantum mechanics/molecular mechanics approach to investigate the reaction mechanism of TDG-catalyzed N-glycosidic bond cleavage of 5-caC. The calculated results show that TDG-catalyzed 5-caC excision follows a concerted (SN2) mechanism in which glycosidic bond dissociation is coupled with nucleophile attack. Protonation of the 5-caC anion contributes to the cleavage of the N-glycoside bond, in which the N3-protonated zwitterion and imino tautomers are more favorable than carboxyl-protonated amino tautomers. This is consistent with the experimental data. Furthermore, our results reveal that the configuration rearrangement process of the protonated 5-caC would lower the stability of the N-glycoside bond and substantially reduce the barrier height for the subsequent C1'-N1 bond cleavage. This should be attributed to the smaller electrostatic repulsion between the leaving base and the negative phosphate group as a result of the structural rearrangement.