An α-ketoglutarate conformational switch controls iron accessibility, activation, and substrate selection of the human FTO protein.

The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N⁶-methyladenosine (m⁶A), N¹-methyladenosine (m¹A), 3-methylthymine (m³T), and 3-methyluracil (m³U) in single-stranded nucleic acids. It is well established that the catalytic activity of FTO proceeds via two coupled reactions. The first reaction involves decarboxylation of alpha-ketoglutarate (αKG) and formation of an oxyferryl species. In the second reaction, the oxyferryl intermediate oxidizes the methylated nucleic acid to reestablish Fe(II) and the canonical base. However, it remains unclear how binding of the nucleic acid activates the αKG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5-mer DNA oligos incorporating the m⁶A, m¹A, or m³T modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of the nucleic acid to FTO activates a two-state conformational equilibrium in the αKG cosubstrate that modulates the O₂ accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the αKG conformation in which Fe(II) is exposed to O₂ are demethylated more efficiently by FTO. These results indicate that i) binding of the methylated nucleic acid is required to expose the catalytic metal to O₂ and activate the αKG decarboxylation reaction, and ii) the measured turnover of the demethylation reaction (which is an ensemble average over the entire sample) depends on the ability of the methylated base to favor the Fe(II) state accessible to O₂.