Computations Reveal a Rich Mechanistic Variation of Demethylation of N-Methylated DNA/RNA Nucleotides by FTO

The fat-mass and obesity-associated (FTO) protein employs an iron(IV) oxo species to demethylate N-methylated nucleic acids. Herein, we use atomistic-theoretical calculations to study the demethylation of the N-methylated DNA/RNA bases 6-methylated adenine (m⁶A), 3-methylated thymine (m³T), and 3-methylated uracil (m³U). The mechanisms involve in-enzyme hydroxylation of the methyl group, followed by hydrolysis of the oxidized intermediates in aqueous solution to demethylate the bases. The in-enzyme reactions have been studied using quantum mechanical/molecular mechanical (QM/MM) calculations, while the hydrolytic reactions occurring outside the enzyme have been explored with hybrid cluster-continuum (HCC) calculations. When the results obtained with these different methods are combined, the calculated barrier for the overall transformation is consistent with the experimental free energy barrier for the major route of m⁶A demethylation: in this pathway, adenine's N1 site acts as an internal base catalyst in the rate-determining hydrolysis of the hydroxylated hemiaminal intermediate hm⁶A to a demethylated A and formaldehyde. This N1-catalyzed mechanism makes m⁶A the most reactive substrate in comparison to other bases we tested. In the minor, slower, route, two oxidation steps by FTO generate an amide intermediate (f⁶A) that undergoes in-water hydrolysis, producing A and formic acid, as found experimentally. In contrast, since m³T and m³U lack internal basic catalytic sites, their hemiaminals decompose with high barriers. The mechanism instead involves two sequential oxidations, leading to demethylated bases and formic acid. Thus, our results, obtained using a holistic approach combining modeling the enzyme and the surrounding aqueous solution, suggest revisions of the experimental mechanisms for m³T and m³U demethylation.