Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates.

摘要: The AlkB family of nonheme Fe(II)/2-oxoglutarate-dependent oxygenases are essential regulators RNA epigenetics by serving as erasers one-carbon marks on with release formaldehyde (FA). Two major human members, FTO and ALKBH5, both act oxidative demethylases N6-methyladenosine (m6A) but furnish different products, N6-hydroxymethyladenosine (hm6A) adenosine (A), respectively. Here we identify foundational mechanistic differences between ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional N-demethylation pathway catalyze conversion m6A hm6A subsequent slow A FA, find catalyzes direct m6A-to-A transformation rapid FA release. We catalytic R130/K132/Y139 triad within facilitates via an unprecedented covalent-based demethylation mechanism detection covalent intermediate. Importantly, K132Q mutant furnishes enzyme profile resembles establishing the importance this residue in proposed mechanism. Finally, show is endogenous source cell activity-based sensing fluxes perturbed knockdown. This work provides fundamental rationale for nonredundant roles beyond substrate preferences cellular localization, where versus results unit potential genotoxin, at rates living systems.