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 acidcatalyzed and proceeds through an oxocarbenium-like transition state. It also provides insights into the catalytic roles of the Thr197 and Asn140 residues.
Uracil DNA glycosylase catalyzes the N-glycosidic bond cleavage of uracil, thereby initiating the base excision repair mechanism for this DNA lesion. Here we employ hybrid quantum mechanics/molecular mechanics calculations to investigate the exact mechanism of the nucleophile attack and the role of the conserved His148 residue. Our calculations suggest that the C1′−N1 bond dissociation proceeds by a migration of the electrophilic sugar in the direction of the water nucleophile, resulting in a planar, oxocarbenium-like transition state. The subsequent nucleophile addition and proton transfer to a nearby base occur without a barrier. We assign the role of a proton acceptor to His148 and elucidate why mutations of this residue curtail the enzymatic activity but do not fully suppress it.
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