biostudies-literatureCherilakkudy FHNIGMS NIH HHS102779https://www.ebi.ac.uk/biostudies/studies/S-EPMC12439684biostudies-literatureUnknown6(8)ALKBH5 is one of only two known human non-heme Fe(II)/2-oxoglutarate-dependent oxygenases that catalyze the demethylation of N⁶-methyladenine (m⁶A) in single-stranded mRNA, underscoring its role in diverse cancers. Unlike its homolog, the fat mass and obesity-associated protein (FTO), which oxidizes m⁶A to a stable N⁶-hydroxymethyladenine (hm⁶A) intermediate, ALKBH5 demethylates m⁶A, 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 favorCell reports. Physical scienceRevealing the catalytic mechanism of the Fe(II)/2-oxoglutarate-dependent human epigenetic modifying enzyme ALKBH5.PMC12439684R35 GM156437Waheed SOLi DChristov CZCherilakkudy FHThomas MGVenditti VKarabencheva-Christova TGKrishnan AVarghese ASchofield CJfalseRevealing the catalytic mechanism of the Fe(II)/2-oxoglutarate-dependent human epigenetic modifying enzyme ALKBH5.ALKBH5 is one of only two known human non-heme Fe(II)/2-oxoglutarate-dependent oxygenases that catalyze the demethylation of N⁶-methyladenine (m⁶A) in single-stranded mRNA, underscoring its role in diverse cancers. Unlike its homolog, the fat mass and obesity-associated protein (FTO), which oxidizes m⁶A to a stable N⁶-hydroxymethyladenine (hm⁶A) intermediate, ALKBH5 demethylates m⁶A, 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 favor2025-01-01T00:00:00Z2025 Aug2026-06-01T06:21:59.503Z2026-04-08T09:46:29.097ZS-EPMC124396844096369310.1016/j.xcrp.2025.102779