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

Abstract

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.

Preorganized Internal Electric Field Powers Catalysis in the Active Site of Uracil-DNA Glycosylase

“…A transition state (TS1) is identified with a Gibbs free energy barrier of 6.5 kcal/mol, where the glycosidic bond has been stretched to 2.25 Å and the nucleophile distance shortened to 2.59 Å. The electronic energy of TS1 is 1.1 kcal/mol higher than the corresponding Gibbs free energy, indicating a small entropic effect during this step which is consistent with experimental results 100 as well as computational studies 85,101,102 of related DNA glycosylases. The accumulated mulliken charge on uracil ring has increased to 0.71 e − .…”

“…The hybrid B3LYP − density functional with two levels of theory was applied to the QM region, while the remaining MM region was treated with the Amber parameters. B3LYP was chosen due to its wide application in the studies of DNA glycosylases. ,− ,− Transition states were located with relaxed potential energy surface scans followed by full TS optimizations using the dimer optimizer implemented in the DL-FIND code . For geometry optimization and scanning calculations, a small basis set of 6-31G­(d) was used for all atoms (labeled as B1).…”

DNA Deformation Exerted by Regulatory DNA-Binding Motifs in Human Alkyladenine DNA Glycosylase Promotes Base Flipping

“…How AAG recognizes the rare lesions among a vast amount of cognate nucleotides is an intriguing question endeavored by extensive experimental/theoretical studies. − Early efforts among these have established a facilitated diffusion mechanism for target searching: AAG first encounters a non-specific site on the DNA via random 3D diffusion and then finds its target lesion via short-range sliding/hopping along the DNA chain. − AAG, like many DNA glycosylases and DNA repairing/modifying proteins, adopts a base-flipping strategy to extrude the target nucleotide from the DNA duplex and catalyze the flipped nucleobase in its active site. − However, the role of DNA glycosylases in such a flipping process remains debatable. Former nuclear magnetic resonance spectroscopy studies indicate that uracil DNA glycosylase acts passively to trap the extrahelical nucleotide that can spontaneously swing out of DNA duplex prior to uracil DNA glycosylase binding. , By contrast, active involvements in the base-flipping process have been proposed for several other DNA glycosylases.…”

Computational studies of DNA repair: Insights into the function of monofunctional DNA glycosylases in the base excision repair pathway

“…AAG was also determined to be able to use the chemical step to discriminate against canonical A, with differential binding in the AAG active site resulting in nucleoside misalignment and poor nucleophile water activation. Subsequent QM/MM (ONIOM(B3LYP/6‐31G(d):AMBER)) calculations with the bound nucleotide and key active site residues in the high‐level region (Glu125, Tyr127, His136, Tyr159, and Asn169, as well as active site water) confirmed a similar mechanistic story for the Hx, 7MeG and G series . Specifically, excision of Hx was found to be facilitated by π–π interactions and hydrogen bonds with active site water, while disruption of the Glu125 base due to misalignment of canonical G in the active site was predicted to prohibit glycosidic bond cleavage.…”