Reaction Mechanism for the N-Glycosidic Bond Cleavage of 5-Formylcytosine by Thymine DNA Glycosylase.

Thymine DNA glycosylase (TDG) initiates the base excision repair mechanism for the deamination and oxidation products of cytosine and 5-methylcytosine. This enzyme has a key role in epigenetic regulation, and its catalytic inactivation results in, e.g., mice embryo lethality. Here, we employ molecular dynamics simulations and quantum mechanics/molecular mechanics calculations to investigate the reaction mechanism of the TDG-catalyzed N-glycosidic bond hydrolysis of the modified base 5-formylcytosine. Our results reveal a reaction pathway, which in its first step features a reorganization of the substrate that lowers the barrier height for the subsequent C1'-N1 bond dissociation. The suggested mechanism is consistent with the experimental data, as it is not acid-catalyzed and proceeds through an oxocarbenium-like transition state. It also provides insights into the catalytic roles of the Thr197 and Asn140 residues.