Zhao Yuan, Chen Nanhao, Mo Yirong, Cao Zexing
State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China.
Phys Chem Chem Phys. 2014 Dec 28;16(48):26864-75. doi: 10.1039/c4cp04032e.
Hydroxynitrile lyases (HNLs) defend plants from herbivores and microbial attack by releasing cyanide from hydroxynitriles. The reverse process has been productively applied to bioorganic syntheses of pharmaceuticals and agrochemicals. To improve our understanding of the catalytic mechanism of HNLs, extensive ab initio QM/MM and classical MM molecular dynamics simulations have been performed to explore the catalytic conversion of cyanohydrins into aldehyde (or ketone) and HCN by hydroxynitrile lyases from Hevea brasiliensis (HbHNLs). It was found that the catalytic reaction approximately follows a two-stage mechanism. The first stage involves two fast processes including the proton abstraction of the substrate through a double-proton transfer and the C-CN bond cleavage, while the second stage concerns HCN formation and is rate-determining. The complete free energy profile exhibits a peak of ∼18 kcal mol(-1). Interestingly, the protonation state of Lys236 influences the efficiency of the enzyme only to some extent, but it changes the entire catalytic mechanism. The dynamical behaviors of substrate delivery and HCN release are basically modulated by the gate movement of Trp128. The remarkable exothermicity of substrate binding and the facile release of HCN may drive the enzyme-catalyzed reaction to proceed along the substrate decomposition efficiently. Computational mutagenesis reveals the key residues which play an important role in the catalytic process.
醇腈酶（HNLs）通过从羟基腈中释放氰化物来保护植物免受食草动物和微生物的攻击。相反的过程已被有效地应用于药物和农用化学品的生物有机合成。为了更好地理解HNLs的催化机制，我们进行了广泛的从头算量子力学/分子力学（QM/MM）和经典分子力学（MM）分子动力学模拟，以探索巴西橡胶树（HbHNLs）中的醇腈酶将氰醇催化转化为醛（或酮）和HCN的过程。结果发现，催化反应大致遵循两阶段机制。第一阶段包括两个快速过程，即通过双质子转移对底物进行质子抽取和C-CN键断裂，而第二阶段涉及HCN的形成，是速率决定步骤。完整的自由能剖面图显示有一个约18千卡/摩尔（-1）的峰值。有趣的是，Lys236的质子化状态仅在一定程度上影响酶的效率，但它改变了整个催化机制。底物传递和HCN释放的动力学行为基本上由Trp128的门控运动调节。底物结合的显著放热性和HCN的容易释放可能驱动酶催化反应沿着底物分解有效地进行。计算诱变揭示了在催化过程中起重要作用的关键残基。
