Insights into the Glycyl Radical Enzyme Active Site of Benzylsuccinate Synthase: A Computational Study,Journal of the American Chemical Society

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Insights into the Glycyl Radical Enzyme Active Site of Benzylsuccinate Synthase: A Computational Study
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2013-08-07 , DOI: 10.1021/ja404842r
Vivek S. Bharadwaj ₁ , Anthony M. Dean ₁ , C. Mark Maupin ₁

Affiliation

The fumarate addition reaction, catalyzed by the enzyme benzylsuccinate synthase (BSS), is considered to be one of the most intriguing and energetically challenging reactions in biology. BSS belongs to the glycyl radical enzyme family and catalyzes the fumarate addition reaction, which enables microorganisms to utilize hydrocarbons as an energy source under anaerobic conditions. Unfortunately, the extreme sensitivity of the glycyl radical to oxygen has hampered the structural and kinetic characterization of BSS, thereby limiting our knowledge on this enzyme. To enhance our molecular-level understanding of BSS, a computational approach involving homology modeling, docking studies, and molecular dynamics (MD) simulations has been used to deduce the structure of BSS's catalytic subunit (BSSα) and illuminate the molecular basis for the fumarate addition reaction. We have identified two conserved and distinct binding pockets at the BSSα active site: a hydrophobic pocket for toluene binding and a polar pocket for fumaric acid binding. Subsequent dynamical and energetic evaluations have identified Glu509, Ser827, Leu390, and Phe384 as active site residues critical for substrate binding. The orientation of substrates at the active site observed in MD simulations is consistent with experimental observations of the syn addition of toluene to fumaric acid. It is also found that substrate binding tightens the active site and restricts the conformational flexibility of the thiyl radical, leading to hydrogen transfer distances conducive to the proposed reaction mechanism. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition.

中文翻译：

对苄基琥珀酸合酶的甘氨酰自由基酶活性位点的深入了解：一项计算研究

由苄基琥珀酸合成酶 (BSS) 催化的延胡索酸加成反应被认为是生物学中最有趣和最具挑战性的反应之一。BSS属于甘氨酰自由基酶家族，催化富马酸加成反应，使微生物能够在厌氧条件下利用碳氢化合物作为能源。不幸的是，甘氨酰自由基对氧的极端敏感性阻碍了 BSS 的结构和动力学表征，从而限制了我们对这种酶的了解。为了增强我们对 BSS 的分子级理解，一种涉及同源建模、对接研究和分子动力学 (MD) 模拟的计算方法已被用于推断 BSS 的结构 s 催化亚基 (BSSα) 并阐明富马酸盐加成反应的分子基础。我们在 BSSα 活性位点确定了两个保守且不同的结合口袋：一个用于甲苯结合的疏水口袋和一个用于富马酸结合的极性口袋。随后的动态和能量评估已将 Glu509、Ser827、Leu390 和 Phe384 确定为对底物结合至关重要的活性位点残基。在 MD 模拟中观察到的活性位点底物的取向与甲苯向富马酸顺式加成的实验观察结果一致。还发现底物结合收紧了活性位点并限制了硫自由基的构象灵活性，导致氢转移距离有利于所提出的反应机制。

更新日期：2013-08-07

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