Document Type

Article

Publication Date

7-27-2026

Department

Department of Chemistry; Department of Biomedical Engineering; Department of Biological Sciences

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-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 , N -methyl ethyl lysine () and N -isopropyl lysine (). 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.

Publisher's Statement

© 2026 The Authors. Published by American Chemical Society. Publisher’s version of record: https://doi.org/10.1021/jacsau.6c00575

Publication Title

JACS Au

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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