Decoding the Catalytic Strategy for DNA Alkylation Repair by AlkA: Insights from MD Simulations and QM/MM Calculations

DNA is under constant attack by exogenous and endogenous agents that result in alkylation damage, which can lead to mutagenesis and cell death. 3-methyladenine DNA glycosylase II (AlkA) initiates the repair of many alkylated nucleotides in bacterial cells by cleaving the glycosidic bond connecting the damaged nucleobase to deoxyribose. However, due to the lack of a crystal structure for the enzyme with a substrate nucleotide bound in the active site, it is poorly understood how AlkA accommodates such a wide variety of substrates and there is controversy regarding the AlkA catalytic mechanism. The present study uses MD simulations to investigate AlkA interactions with DNA containing a major substrate, 3-methyladenine (3mA), and QM/MM calculations to map multiple potential pathways for 3mA excision. MD simulations reveal eight residues comprise the AlkA active site, binding the substrate through several non-specific, hydrophobic contacts. Substrate promiscuity is uncovered as likely to be further promoted by Y273, which can change conformation to modulate the binding pocket size. Despite a previous proposal that AlkA actvitity involves DNA−protein crosslink formation, QM/MM calculations suggest that catalysis proceeds through a direct hydrolysis mechanism in which D238 activates a water nucleophile that attacks at C1′ of the damaged nucleotide. Our combined MD and QM/MM approach highlights that proper D238 positioning with respect to the substrate is supported by a network of D238 interactions with the substrate, W218, and W272. The proposed mechanism is consistent with experimental kinetic, mutagenic, and structural data for AlkA, and aligns with the preferred pathway of many other monofunctional glycosylases. The insights into AlkA substrate binding and catalysis gained from this study can push forward the design of small molecule inhibitors as antibacterial agents and broaden our general understanding of DNA repair pathways, which can aid in the development of cancer therapeutics.