PHF8 (KDM7B) is a human non-heme 2-oxoglutarate (2OG) JmjC domain oxygenase that catalyzes the demethylation of the di/mono-Nε-methylated K9 residue of histone H3. Altered PHF8 activity is linked to genetic diseases and cancer; thus, it is an interesting target for epigenetic modulation. We describe the use of combined quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulations to explore the mechanism of PHF8, including dioxygen activation, 2OG binding modes, and substrate demethylation steps. A PHF8 crystal structure manifests the 2OG C-1 carboxylate bound to iron in a non-productive orientation, i.e., trans to His247. A ferryl-oxo intermediate formed by activating dioxygen bound to the vacant site in this complex would be non-productive, i.e., ‘off-line’ with respect to reaction with Nε-methylated K9. We show rearrangement of the ‘off-line’ ferryl-oxo intermediate to a productive ‘in-line’ geometry via a solvent exchange reaction (called “ferryl-flip”) is energetically unfavorable. The calculations imply that movement of the 2OG C-1 carboxylate prior to dioxygen binding at a five-coordination stage in catalysis proceeds with a low barrier, suggesting two possible 2OG C-1 carboxylate geometries can coexist at room temperature. We explored alternative mechanisms for hydrogen atom transfer and show that second sphere interactions orient the Nε-methylated lysine in a conformation where hydrogen abstraction from a methyl C-H bond is energetically more favorable than hydrogen abstraction from the N-H bond of the protonated Nε-methyl group. Using multiple HAT reaction path calculations, we demonstrate the crucial role of conformational flexibility in effective hydrogen transfer. Subsequent hydroxylation occurs through a rebound mechanism, which is energetically preferred compared to desaturation, due to second sphere interactions. The overall mechanistic insights reveal the crucial role of iron-center rearrangement, second sphere interactions and conformational flexibility in PHF8 catalysis and provide knowledge useful for the design of mechanism-based PHF8 inhibitors.

AlkB and its human homologue AlkBH2 are Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases that repair alkylated DNA bases occurring as a consequence of reactions with mutagenic agents. We used molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) methods to investigate how structural dynamics influences the selectivity and mechanisms of the AlkB- and AlkBH2-catalyzed demethylation of 3-methylcytosine (m₃C) in single (ssDNA) and double (dsDNA) stranded DNA. Dynamics studies reveal the importance of the flexibility in both the protein and DNA components in determining the preferences of AlkB for ssDNA and of AlkBH2 for dsDNA. Correlated motions, including of a hydrophobic β-hairpin, are involved in substrate binding in AlkBH2–dsDNA. The calculations reveal that 2OG rearrangement prior to binding of dioxygen to the active site Fe is preferred over a ferryl rearrangement to form a catalytically productive Fe(IV)═O intermediate. Hydrogen atom transfer proceeds via a σ-channel in AlkBH2–dsDNA and AlkB–dsDNA; in AlkB–ssDNA, there is a competition between σ- and π-channels, implying that the nature of the complexed DNA has potential to alter molecular orbital interactions during the substrate oxidation. Our results reveal the importance of the overall protein–DNA complex in determining selectivity and how the nature of the substrate impacts the mechanism.

A forward-looking perspective on optimizing enzyme design through synergizing electric fields, coordination spheres, and dynamics.

The N^ε-methyl lysine status of histones is important in the regulation of eukaryotic transcription. The Fe(II) and 2-oxoglutarate (2OG) -dependent JmjC domain enzymes are the largest family of histone N^ε-methyl lysine demethylases (KDMs). The human KDM4 subfamily of JmjC KDMs is linked with multiple cancers and some of its members are medicinal chemistry targets. We describe the use of combined molecular dynamics (MD) and Quantum Mechanical/Molecular Mechanical (QM/MM) methods to study the mechanism of KDM4A, which catalyzes demethylation of both tri- and di-methylated forms of histone H3 at K9 and K36. The results show that the oxygen activation at the active site of KDM4A is optimized towards the generation of the reactive Fe(IV)-oxo intermediate. Factors including the substrate binding mode, correlated motions of the protein and histone substrates, and molecular orbital control synergistically contribute to the reactivity of the Fe(IV)-oxo intermediate. In silico substitutions were performed to investigate the roles of residues (Lys241, Tyr177, and Asn290) in substrate orientation. The Lys241Ala substitution abolishes activity due to altered substrate orientation consistent with reported experimental studies. Calculations with a macrocyclic peptide substrate analogue reveal that induced conformational changes/correlated motions in KDM4A are sequence-specific in a manner that influences substrate binding affinity. Second sphere residues, such as Ser288 and Thr289, may contribute to KDM4A catalysis by correlated motions with active site residues. Residues that stabilize key intermediates, and which are predicted to be involved in correlated motions with other residues in the second sphere and beyond, are shown to be different in KDM4A compared to those in another JmjC KDM (PHF8), which acts on H3K9 di- and mono-methylated forms, suggesting that allosteric type inhibition is of interest from the perspective of developing selective JmjC KDM inhibitors.