Catalysis by the Non-Heme Iron(II) Histone Demethylase PHF8 Involves Iron Center Rearrangement and Conformational Modulation of Substrate Orientation
Catalysis by the Non-Heme Iron(II) Histone Demethylase PHF8 Involves Iron Center Rearrangement and Conformational Modulation of Substrate Orientation
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DOI：
10.1021/acscatal.9b04907
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发表时间：
2020-01-17
期刊：
影响因子：
12.9
通讯作者：
Christov, Christo Z.
中科院分区：
文献类型：
--
作者：
Chaturvedi, Shobhit S.;Ramanan, Rajeev;Christov, Christo Z.
PHF8 （KDM7B）是一种人非血红素2-氧戊二酸（20G） JmjC结构域加氧酶，可催化组蛋白H3的二/单n- epsilon甲基化K9残基的去甲基化。PHF8活性的改变与遗传疾病和癌症有关；因此，它是表观遗传调控的一个有趣的靶点。我们描述了使用量子力学/分子力学（QM/MM）和分子动力学（MD）模拟来探索PHF8的机制，包括双氧活化，2OG结合模式和底物去甲基化步骤。PHF8的晶体结构显示出2OG C-1羧酸盐以非生产性取向与铁结合，即反式到His247。通过激活与该复合体空位点结合的二氧形成的铁酰氧中间体是非生产性的，即与ne -甲基化K9的反应“脱机”。我们表明，通过溶剂交换反应（称为“铁基翻转”）将“离线”铁基氧中间体重排为生产性“在线”几何结构，在能量上是不利的。计算表明，在催化过程中，在五配位阶段，20G C-1羧酸盐在双氧结合之前的运动以低势垒进行，这表明两种可能的20G C-1羧酸盐几何形状可以在室温下共存。我们探索了氢原子转移的其他机制，并表明第二球相互作用使ne -甲基化赖氨酸的构象从甲基C-H键中提取氢比从质子化ne -甲基的N-H键中提取氢在能量上更有利。通过多个HAT反应路径计算，我们证明了构象柔韧性在有效氢转移中的关键作用。随后的羟基化通过反弹机制发生，由于第二球相互作用，与去饱和相比，它在能量上更有利。整体机理揭示了铁中心重排、第二球相互作用和构象灵活性在PHF8催化中的关键作用，并为基于机理的PHF8抑制剂的设计提供了有用的知识。
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.