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

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.