Catalysis by the non-heme iron(II) histone demethylase PHF8 involves iron center rearrangement and conformational modulation of substrate orientation
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...
| Main Authors: | , , , , , |
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| Format: | Journal article |
| Language: | English |
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American Chemical Society
2019
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| _version_ | 1797063061639528448 |
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| author | Chaturvedi, SS Ramanan, R Lehnert, N Schofield, CJ Karabencheva-Christova, TG Christov, CZ |
| author_facet | Chaturvedi, SS Ramanan, R Lehnert, N Schofield, CJ Karabencheva-Christova, TG Christov, CZ |
| author_sort | Chaturvedi, SS |
| collection | OXFORD |
| description | 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 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 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 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 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. |
| first_indexed | 2024-03-06T20:54:28Z |
| format | Journal article |
| id | oxford-uuid:38b6bcc4-c030-4e7d-af2d-d2826a206396 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T20:54:28Z |
| publishDate | 2019 |
| publisher | American Chemical Society |
| record_format | dspace |
| spelling | oxford-uuid:38b6bcc4-c030-4e7d-af2d-d2826a2063962022-03-26T13:51:45ZCatalysis by the non-heme iron(II) histone demethylase PHF8 involves iron center rearrangement and conformational modulation of substrate orientationJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:38b6bcc4-c030-4e7d-af2d-d2826a206396EnglishSymplectic Elements at OxfordAmerican Chemical Society2019Chaturvedi, SSRamanan, RLehnert, NSchofield, CJKarabencheva-Christova, TGChristov, CZPHF8 (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 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 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 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 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. |
| spellingShingle | Chaturvedi, SS Ramanan, R Lehnert, N Schofield, CJ Karabencheva-Christova, TG Christov, CZ Catalysis by the non-heme iron(II) histone demethylase PHF8 involves iron center rearrangement and conformational modulation of substrate orientation |
| title | Catalysis by the non-heme iron(II) histone demethylase PHF8 involves iron center rearrangement and conformational modulation of substrate orientation |
| title_full | Catalysis by the non-heme iron(II) histone demethylase PHF8 involves iron center rearrangement and conformational modulation of substrate orientation |
| title_fullStr | Catalysis by the non-heme iron(II) histone demethylase PHF8 involves iron center rearrangement and conformational modulation of substrate orientation |
| title_full_unstemmed | Catalysis by the non-heme iron(II) histone demethylase PHF8 involves iron center rearrangement and conformational modulation of substrate orientation |
| title_short | Catalysis by the non-heme iron(II) histone demethylase PHF8 involves iron center rearrangement and conformational modulation of substrate orientation |
| title_sort | catalysis by the non heme iron ii histone demethylase phf8 involves iron center rearrangement and conformational modulation of substrate orientation |
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