Beyond Histone Demethylation: Mechanisms of N-Alkyl Consecutive Oxidations by the Non-Heme Fe(II)/2-Oxoglutarate Oxygenase KDM6B

Authors

• Simahudeen Bathir Jaber Sathik Rifayee, Michigan Technological University
• Sudheesh Devadas, Michigan Technological University
• Midhun George Thomas, Michigan Technological University
• Bhargav Varada, Michigan Technological University
• Ethan Sommer, College of Engineering
• Cassandra Talaba, Michigan Technological University
• Christopher Schofield, University of Oxford
• Christo Z. Christov, Michigan Technological University

Document Type

Article

Publication Date

7-27-2026

Abstract

The nonheme Fe(II)/2-oxoglutarate (2OG)-dependent histone demethylase KDM6B (JMJD3) has demonstrated a capacity for diversity in the oxidative transformations of N^ε-alkylated lysine residues in histone H3 peptides; however, the mechanisms of such dealkylations, compared with standard KDM-catalyzed demethylations, remain unexplored. We implemented molecular dynamics and quantum mechanics/molecular mechanics to investigate the catalytic strategies for the sequential oxidation reactions of KDM6B with different N-alkylated forms of lysine K27 in the H3 peptide chain, that is N^ε, N^ε-methyl ethyl lysine (Lys(Me/Eth)) and N^ε-isopropyl lysine (Lys(iPr)). The results for sequential oxidations, which yield alcohol, aldehyde, and then carboxylic acid products, reveal that variations in the conformational positioning of different N-alkylated groups are enabled by second coordination sphere (SCS) interactions and long-range correlated motions. Specifically, access of the different N-alkylated groups to the reactive Fe(IV)=O intermediate, leading to hydroxylation, is controlled by a network of SCS interactions, in particular involving N344 and Y239, which was also demonstrated by MD and QM/MM calculations on N344A and Y239A mutants. Subsequent oxidations of the alcohols to aldehyde and acid derivatives are also guided by the conformational positioning of the hydroxylated/aldehyde substituent. QM/MM calculations predicted regio- and chemo-selective oxidation can be initiated through hydrogen atom transfer involving σ- or π-mechanisms. The insights would guide experimental efforts to design Fe(II)/2OG enzymes with non-native catalytic activities and altered substrate selectivity. Furthermore, the results reveal mechanistic features that can be leveraged to design biocatalytic platforms for the selective functionalization of peptide-based drugs.

Publication Title

Jacs Au

Recommended Citation

Jaber Sathik Rifayee, S., Devadas, S., Thomas, M., Varada, B., Sommer, E., Talaba, C., Schofield, C., & Christov, C. (2026). Beyond Histone Demethylation: Mechanisms of N-Alkyl Consecutive Oxidations by the Non-Heme Fe(II)/2-Oxoglutarate Oxygenase KDM6B. Jacs Au, 6(7), 4048-4072. http://doi.org/10.1021/jacsau.6c00575
Retrieved from: https://digitalcommons.mtu.edu/michigantech-p2/2813