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Catalysis by the Non-Heme Iron(II) Histone Demethylase PHF8 Involves Iron Center Rearrangement and Conformational Modulation of Substrate Orientation

[Image: see text] PHF8 (KDM7B) is a human non-heme 2-oxoglutarate (2OG) JmjC domain oxygenase that catalyzes the demethylation of the di/mono-N(ε)-methylated K9 residue of histone H3. Altered PHF8 activity is linked to genetic diseases and cancer; thus, it is an interesting target for epigenetic mod...

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Detalles Bibliográficos
Autores principales: Chaturvedi, Shobhit S., Ramanan, Rajeev, Lehnert, Nicolai, Schofield, Christopher J., Karabencheva-Christova, Tatyana G., Christov, Christo Z.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2019
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970271/
https://www.ncbi.nlm.nih.gov/pubmed/31976154
http://dx.doi.org/10.1021/acscatal.9b04907
Descripción
Sumario:[Image: see text] PHF8 (KDM7B) is a human non-heme 2-oxoglutarate (2OG) JmjC domain oxygenase that catalyzes the demethylation of the di/mono-N(ε)-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 nonproductive orientation, i.e., trans to His247. A ferryl–oxo intermediate formed by activating dioxygen bound to the vacant site in this complex would be nonproductive, i.e., “off-line” with respect to reaction with N(ε)-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 that 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 N(ε)-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 N(ε)-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.