Insights into the Glycyl Radical Enzyme Active Site of Benzylsuccinate Synthase: A Computational Study

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.由苄基琥珀酸合酶（benzylsuccinate synthase, BSS）催化的富马酸加成反应，被认为是生物学中最引人入胜且能量学上极具挑战性的反应之一。BSS属于甘氨酰自由基酶（glycyl radical enzyme）家族，可催化该富马酸加成反应，使微生物能够在厌氧条件下以烃类作为能量来源。遗憾的是，甘氨酰自由基对氧气的极端敏感性阻碍了BSS的结构与动力学表征，进而限制了我们对该酶的认知。为加深我们对BSS的分子层面理解，本研究采用了包含同源建模、分子对接研究以及分子动力学（MD）模拟在内的计算方法，以推导BSS催化亚基（BSSα）的结构，并阐明富马酸加成反应的分子基础。我们在BSSα的活性位点中鉴定出两个保守且独特的结合口袋：一个用于结合甲苯的疏水口袋，以及一个用于结合富马酸的极性口袋。后续的动力学与能量学评估鉴定出Glu509、Ser827、Leu390与Phe384为对底物结合至关重要的活性位点残基。MD模拟中观测到的底物在活性位点的取向，与甲苯与富马酸发生顺式加成的实验观测结果一致。研究还发现，底物结合会收紧活性位点并限制硫自由基（thiyl radical）的构象灵活性，使得氢转移距离符合所提出的反应机制。底物在活性位点的稳定性，以及硫自由基、底物与活性位点甘氨酸之间存在可行的自由基转移距离，共同表明存在一条由底物辅助、调控富马酸加成反应的自由基转移途径。