"Cracking the AlkB Homologues' Code: How an α-ketoglutarate conformational equilibrium controls iron accessibility, activation and substrate selection"

Abstract: The AlkB family of dioxygenases regulates a myriad of cellular processes by catalyzing the oxidative demethylation of methyl modified proteins and nucleic acids 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 or protein substrate to reestablish Fe(II) and the canonical base or amino acid. Given their central metabolic role, extensive effort has gone into identifying the catalytic mechanism and physiological substrates of AlkB enzymes. Yet it is still unclear how binding of the primary substrate activates the αKG decarboxylation reaction, and why AlkB enzymes processes different methyl modifications with different turnover rates. Here, we investigate the interaction of FTO and Alkbh5, two nucleic acid demethylates members of the Alkb family, with 5-mer DNA oligos incorporating the N6-methyladenosine (m⁶A), N1-methyladenosine (m¹A), and 3-methylthymine (m³T) modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of αKG and DNA progressively quenches conformational dynamics within the catalytic domain, therefore explaining the orchestrated sequential binding of αKG and primary substrate, which is an hallmark of AlkB enzymes. In addition, we show that binding of the nucleic acid to the enzyme activates a two-state conformational equilibrium in the αKG co-substrate 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 or Alkbh5. 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 (Purslow et al Sci. Adv. 2021 7: eabi8215; Burns et al Proc. Natl. Acad. Sci. USA 2024 121: e2404457121). Since all AlkB enzymes catalyze similar reactions on similar substrates, it is plausible that the activation and substrate selectivity mechanisms elucidated here are shared among other members of the AlkB family.