How Do Electrostatic Perturbations of the Protein Affect the Bifurcation Pathways of Substrate Hydroxylation versus Desaturation in the Nonheme Iron-Dependent Viomycin Biosynthesis Enzyme?
How Do Electrostatic Perturbations of the Protein Affect the Bifurcation Pathways of Substrate Hydroxylation versus Desaturation in the Nonheme Iron-Dependent Viomycin Biosynthesis Enzyme?
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DOI：
10.1021/acs.jpca.1c00141
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发表时间：
2021-02-23
影响因子：
2.9
通讯作者：
de Visser, Sam P.
中科院分区：
文献类型：
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作者：
Ali, Hafiz Saqib;Henchman, Richard H.;de Visser, Sam P.
紫霉素生物合成酶VioC是一种非血红素铁和α-酮戊二酸依赖性双加氧酶，参与抗生素生物合成C-3位L-精氨酸的选择性羟基化。有趣的是，实验研究表明，使用底物类似物，即L-高精氨酸，获得了源自C-3羟基化、C-4羟基化和C-3-C-4去饱和的产物的混合物。为了理解在底物中添加一个CH2基团如何导致选择性和活性的巨大变化，我们决定使用量子力学（QM）簇模型进行计算研究。我们建立了一个大的活性位点簇模型的245个原子，其中包括氧化剂与其第一和第二配位球的影响，以及基板结合口袋。该模型进行了验证，对相关酶和以前的计算研究的文献中的实验工作。此后，针对含有L-Arg的模型和以L-homo-Arg为底物的模型，研究了产生产物和副产物的可能途径。计算的活化自由能预测的产品分布，匹配的实验观察，并给出一个低能量的C-3-羟基化途径的L-精氨酸，而L-高精氨酸，几个障碍被发现是接近的能量，导致产品的混合物。然后，我们使用热化学，价键，和静电模型分析了产品分布的差异的起源。我们的研究表明，L-Arg和L-homo-Arg的C-3-H和C-4-H键强度相似;然而，来自蛋白质的诱导电场的外部扰动显著影响L-Arg的相对C-H键强度，使C-3-H键最弱，并将反应引导到选择性C-3-羟基化通道。因此，蛋白质中的电荷分布和VioC活性位点的诱导电偶极场引导L-Arg底物活化为C-3-羟基化，并且不利于C-4-羟基化途径，而这对于L-homo-Arg不会发生。紧密的底物定位和VioC中第二配位球残基的静电扰动也导致L-Arg的总体反应较慢;然而，它们能够实现高底物选择性。我们的研究强调了蛋白质中第二配位圈的重要性，它定位底物和氧化剂、扰乱电荷分布并实现底物选择性。
The viomycin biosynthesis enzyme VioC is a nonheme iron and alpha-ketoglutarate-dependent dioxygenase involved in the selective hydroxylation of L-arginine at the C-3 position for antibiotics biosynthesis. Interestingly, experimental studies showed that using the substrate analogue, namely, L-homoarginine, a mixture of products was obtained originating from C-3 hydroxylation, C-4-hydroxylation, and C-3-C-4-desaturation. To understand how the addition of one CH2 group to a substrate can lead to such a dramatic change in selectivity and activity, we decided to perform a computational study using quantum mechanical (QM) cluster models. We set up a large active-site cluster model of 245 atoms that includes the oxidant with its first-and second-coordination sphere influences as well as the substrate binding pocket. The model was validated against experimental work from the literature on related enzymes and previous computational studies. Thereafter, possible pathways leading to products and byproducts were investigated for a model containing L-Arg and one for L-homo-Arg as substrate. The calculated free energies of activation predict product distributions that match the experimental observation and give a low-energy C-3-hydroxylation pathway for L-Arg, while for L-homo-Arg, several barriers are found to be close in energy leading to a mixture of products. We then analyzed the origins of the differences in product distributions using thermochemical, valence bond, and electrostatic models. Our studies show that the C-3-H and C-4-H bond strengths of L-Arg and L-homo-Arg are similar; however, external perturbations from an induced electric field of the protein affect the relative C-H bond strengths of L-Arg dramatically and make the C-3-H bond the weakest and guide the reaction to a selective C-3-hydroxylation channel. Therefore, the charge distribution in the protein and the induced electric dipole field of the active site of VioC guides the L-Arg substrate activation to C-3-hydroxylation and disfavors the C-4-hydroxylation pathway, while this does not occur for L-homo-Arg. Tight substrate positioning and electrostatic perturbations from the second-coordination sphere residues in VioC also result in a slower overall reaction for L-Arg; however, they enable a high substrate selectivity. Our studies highlight the importance of the second-coordination sphere in proteins that position the substrate and oxidant, perturb charge distributions, and enable substrate selectivity.