Gillet N, Ruiz-Pernía J J, de la Lande A, Lévy B, Lederer F, Demachy I, Moliner V
Laboratoire de Chimie-Physique, CNRS UMR 8000, Université Paris Sud, Bâtiment 349 - Campus d'Orsay. 15, avenue Jean Perrin, 91405 Cedex Orsay, France.
Phys Chem Chem Phys. 2016 Jun 21;18(23):15609-18. doi: 10.1039/c6cp00395h. Epub 2016 May 25.
In this work, we have performed molecular dynamics simulations using a hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) scheme to study the mechanism of l-lactate oxidation by flavocytochrome b2 (Fcb2). Our results obtained at the QM(AM1)/MM level have been improved by single-point corrections using density functional theory (DFT) methods. Free energy surfaces have been calculated in the framework of the hydride transfer hypothesis. This mechanism involves the transfer of the lactate hydroxyl proton to H373 while the substrate αH atom is transferred as a hydride to the flavin mononucleotide (FMN) prosthetic group anchored in the active site. Four different systems have been modeled: wild-type enzyme considering R289 in a distal or a proximal conformation observed in crystal structures and the D282N and Y254L variants (with R289 in a distal position). Simulation results highlight the influence of the environment on the catalytic mechanism by describing a step-wise process in the WT enzyme with R289 in a distal position and a concerted mechanism for the other systems. In the step-wise mechanism, the hydride transfer to flavin can occur only after a proton transfer from substrate to H373. Modifications of the electrostatic field around l-lactate or H373 disfavor the highly charged complex resulting from this proton transfer. Simulations of the Y254L variant also reveal some effect of steric changes.
在这项工作中，我们使用量子力学/分子力学（QM/MM）混合方案进行了分子动力学模拟，以研究黄素细胞色素b2（Fcb2）催化L-乳酸氧化的机制。我们在QM(AM1)/MM水平上获得的结果通过使用密度泛函理论（DFT）方法的单点校正得到了改进。在氢化物转移假说的框架内计算了自由能表面。该机制涉及乳酸羟基质子转移至H373，同时底物αH原子作为氢化物转移至锚定在活性位点的黄素单核苷酸（FMN）辅基。我们对四种不同的体系进行了建模：考虑晶体结构中观察到的处于远端或近端构象的R289的野生型酶，以及D282N和Y254L变体（R289处于远端位置）。模拟结果通过描述R289处于远端位置的野生型酶中的逐步过程以及其他体系的协同机制，突出了环境对催化机制的影响。在逐步机制中，只有在质子从底物转移至H373之后，氢化物才能转移至黄素。L-乳酸或H373周围静电场的改变不利于这种质子转移产生的高电荷复合物。Y254L变体的模拟还揭示了空间变化的一些影响。
