Abstract
ALKBH5 is one of only two known human non-heme Fe(II)/2-oxoglutarate-dependent oxygenases that catalyze the demethylation of N(6)-methyladenine (m(6)A) in single-stranded mRNA, underscoring its role in diverse cancers. Unlike its homolog, the fat mass and obesity-associated protein (FTO), which oxidizes m(6)A to a stable N(6)-hydroxymethyladenine (hm(6)A) intermediate, ALKBH5 demethylates m(6)A, yielding adenine and formaldehyde as products. Here, we integrate molecular dynamics simulations and quantum mechanics/molecular mechanics methods to elucidate ALKBH5's complete catalytic mechanism. Two post-hydroxylation pathways were evaluated: a proton transfer pathway and a Schiff base formation pathway, with the former emerging as the favored mechanism. We identify second-sphere residues Lys132 and Tyr139 as essential contributors to catalysis and demonstrate how Val191 and Tyr133 modulate activity. Dynamic analyses reveal that correlated motions of structural elements such as nucleotide recognition lids NRL1 and NRL2 and increased flexibility of the NRL2 loop in the hm(6)A intermediate may be critical for efficient demethylation.