Journal
ACS CATALYSIS
Volume 10, Issue 2, Pages 1195-1209Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.9b04907
Keywords
histone lysine demethylation; 2-oxoglutarate; non-heme iron enzyme; QM/MM; metal-center rearrangement; enzyme mechanism
Categories
Funding
- Michigan Tech Graduate Teaching Assistantship
- Michigan Tech start-up grants
- NSF [1904215]
- Cancer Research UK [C8717/A18245]
- Wellcome Trust [091857/7/10/7]
- BBSRC [BB/R000344/1] Funding Source: UKRI
- Division Of Chemistry
- Direct For Mathematical & Physical Scien [1904215] Funding Source: National Science Foundation
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PHF8 (KDM7B) is a human non-heme 2-oxoglutarate (20G) JmjC domain oxygenase that catalyzes the demethylation of the di/mono-N-epsilon-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 NE-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 20G 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 NE-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 NE-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.
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