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

• 成果类型: Article
• 署名作者: Burns, Daniel; Khatiwada, Balabhadra; Singh, Aayushi; Purslow, Jeffrey A.; Potoyan, Davit A.; Venditti, Vincenzo
• 署名单位: Iowa State University; Iowa State University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-13254
• DOI: 10.1073/pnas.2404457121
• 发表日期: 2024-06-18
• 关键词: oxidative demethylation; rna demethylase; dna; DYNAMICS; repair; n6-methyladenosine; parameterization; translation; methylation; mechanisms

摘要

The fat mass and obesity- associated fatso (FTO) protein is a member of the Alkb family 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 (alpha KG) 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 alpha KG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5- mer 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 alpha KG cosubstrate that modulates the O2 accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the alpha KG 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 alpha KG 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.