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QM/MM Study of the Reaction Catalyzed by Alkyladenine DNA Glycosylase: Examination of the Substrate Specificity of a DNA Repair Enzyme

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journal contribution
posted on 17.11.2017, 00:00 by Stefan A. P. Lenz, Stacey D. Wetmore
Human alkyladenine DNA glycosylase (AAG) functions as part of the base excision repair pathway to excise structurally diverse oxidized and alkylated DNA purines. Specifically, AAG uses a water molecule activated by a general base and a nonspecific active site lined with aromatic residues to cleave the N-glycosidic bond. Despite broad substrate specificity, AAG does not target the natural purines (adenine (A) and guanine (G)). Using the ONIOM­(QM:MM) methodology, we provide fundamental atomic level details of AAG bound to DNA-containing a neutral substrate (hypoxanthine (Hx)), a nonsubstrate (G), or a cationic substrate (7-methylguanine (7MeG)) and probe changes in the reaction pathway that occur when AAG targets different nucleotides. We reveal that subtle differences in protein–DNA contacts upon binding different substrates within the flexible AAG active site can significantly affect the deglycosylation reaction. Notably, we predict that AAG excises Hx in a concerted mechanism that is facilitated through correct alignment of the (E125) general base due to hydrogen bonding with a neighboring aromatic amino acid (Y127). Hx departure is further stabilized by π–π interactions with aromatic amino acids and hydrogen bonds with active site water. Despite possessing a similar structure to Hx, G is not excised since the additional exocyclic amino group leads to misalignment of the general base due to disruption of the key E125–Y127 hydrogen bond, the catalytically unfavorable placement of water within the active site, and weakened π-contacts between aromatic amino acids and the nucleobase. In contrast, cationic 7MeG does not occupy the same position within the AAG active site as G due to steric clashes with the additional N7 methyl group, which results in the correct alignment of the general base and permits nucleobase excision as observed for neutral Hx. Overall, our structural data rationalizes the observed substrate specificity of AAG and contributes to our fundamental understanding of enzymes with flexible active sites and broad substrate specificities.

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