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

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.