Revealing the catalytic mechanism of the Fe(II)/2-oxoglutarate-dependent human epigenetic modifying enzyme ALKBH5

Authors

Fathima Hameed Cherilakkudy, Michigan Technological UniversityFollow
Midhun George Thomas, Michigan Technological UniversityFollow
Ann Varghese, Michigan Technological UniversityFollow
Sodiq Waheed, Michigan Technological UniversityFollow
Anandhu Krishnan, Michigan Technological UniversityFollow
Vincenzo Venditti, Iowa State University
Christopher J. Schofield, University of Oxford
Deyu Li, College of Pharmacy
Christo Christov, Michigan Technological UniversityFollow
Tatyana Karabencheva-Christova, Michigan Technological UniversityFollow

Document Type

Article

Publication Date

8-20-2025

Department

Department of Chemistry

Abstract

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 favored mechanism. We identify second-sphere residues Lys132 and Tyr139 as essential contributors to catalysis and demonstrate how Val191 and Tyr133 modulate activity. Dynamic analyses reveal that correlated motions of structural elements such as nucleotide recognition lids NRL1 and NRL2 and increased flexibility of the NRL2 loop in the hm⁶A intermediate may be critical for efficient demethylation.

Publisher's Statement

© 2025 The Author(s). Published by Elsevier Inc. Publisher’s version of record: https://doi.org/10.1016/j.xcrp.2025.102779

Publication Title

Cell Reports Physical Science

Recommended Citation

Cherilakkudy, F., Thomas, M., Varghese, A., Waheed, S., Krishnan, A., Venditti, V., Schofield, C., Li, D., Christov, C., & Karabencheva-Christova, T. (2025). Revealing the catalytic mechanism of the Fe(II)/2-oxoglutarate-dependent human epigenetic modifying enzyme ALKBH5. Cell Reports Physical Science, 6(8). http://doi.org/10.1016/j.xcrp.2025.102779
Retrieved from: https://digitalcommons.mtu.edu/michigantech-p2/1941

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.

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