KDM6A (UTX) and KDM6B (JMJD3) are human nonheme Fe(II) and 2-oxoglutarate (2OG) dependent JmjC oxygenases that catalyze the demethylation of trimethylated lysine 27 in the N-terminal tail of histone H3, a post-translational modification that regulates transcription. A Combined Quantum Mechanics/ Molecular Mechanics (QM/MM) and Molecular Dynamics (MD) study on the catalytic mechanism of KDM6A/B reveals that the transition state for the ratelimiting hydrogen atom transfer (HAT) reaction in KDM6A catalysis is stabilized by polar (Asn217) and aromatic (Trp369)/non-polar (Pro274) residues in contrast to KDM4, KDM6B and KDM7 demethylases where charged residues (Glu, Arg, Asp) are involved. KDM6A employs both σand π-electron transfer pathways for HAT, whereas KDM6B employs the σ-electron pathway. Differences in hydrogen bonding of the Fe-chelating Glu252(KDM6B) contribute to the lower energy barriers in KDM6B vs. KDM6A. The study reveals a dependence of the activation barrier of the rebound hydroxylation on the FeÀ OÀ C angle in the transition state of KDM6A. Anti-correlation of the Zn-binding domain with the active site residues is a key factor distinguishing KDM6A/B from KDM7/ 4s. The results reveal the importance of communication between the Fe center, second coordination sphere, and long-range interactions in catalysis by KDMs and, by implication, other 2OG oxygenases.
This study investigates dioxygen binding and 2‐oxoglutarate (2OG) coordination by two model non‐heme FeII/2OG enzymes: a class 7 histone demethylase (PHF8) that catalyzes the hydroxylation of its H3K9me2 histone substrate leading to demethylation reactivity and the ethylene‐forming enzyme (EFE), which catalyzes two competing reactions of ethylene generation and substrate l‐Arg hydroxylation. Although both enzymes initially bind 2OG by using an off‐line 2OG coordination mode, in PHF8, the substrate oxidation requires a transition to an in‐line mode, whereas EFE is catalytically productive for ethylene production from 2OG in the off‐line mode. We used classical molecular dynamics (MD), quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM metadynamics (QM/MM‐MetD) simulations to reveal that it is the dioxygen binding process and, ultimately, the protein environment that control the formation of the in‐line FeIII‐OO⋅− intermediate in PHF8 and the off‐line FeIII‐OO⋅− intermediate in EFE.
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