Max-Planck-Institut für Kohlenforschung , Kaiser-Wilhelm-Platz 1, 45470 Mülheim, Germany.
J Am Chem Soc. 2013 Sep 11;135(36):13400-13. doi: 10.1021/ja403582u. Epub 2013 Aug 29.
We report classical molecular dynamics (MD) simulations and combined quantum mechanics/molecular mechanics (QM/MM) calculations to elucidate the catalytic mechanism of the rate-determining amine oxidation step in the lysine-specific demethylase 1 (LSD1)-catalyzed demethylation of the histone tail lysine (H3K4), with flavin adenine dinucleotide (FAD) acting as cofactor. The oxidation of substrate lysine (sLys) involves the cleavage of an α-CH bond accompanied by the transfer of a hydride ion equivalent to FAD, leading to an imine intermediate. This hydride transfer pathway is shown to be clearly favored for sLys oxidation over other proposed mechanisms, including the radical (or single-electron transfer) route as well as carbanion and polar-nucleophilic mechanisms. MD simulations on six NVT ensembles (covering different protonation states of sLys and K661 as well as the K661M mutant) identify two possible orientations of the reacting sLys and FAD subunits (called "downward" and "upward"). Calculations at the QM(B3LYP-D/6-31G*)/CHARMM22 level provide molecular-level insights into the mechanism, helping to understand how LSD1 achieves the activation of the rather inert methyl-CH bond in a metal-free environment. Factors such as proper alignment of sLys (downward orientation), transition-state stabilization (due to the protein environment and favorable orbital interactions), and product stabilization via adduct formation are found to be crucial for facilitating the oxidative α-CH bond cleavage. The current study also sheds light on the role of important active-site residues (Y761, K661, and W695) and of the conserved water-bridge motif. The steric influence of Y761 helps to position the reaction partners properly, K661 is predicted to get deprotonated prior to substrate binding and to act as an active-site base that accepts a proton from sLys to enable the subsequent amine oxidation, and the water bridge that is stabilized by K661 and W695 mediates this proton transfer.
我们报道了经典分子动力学（MD）模拟和组合量子力学/分子力学（QM/MM）计算，以阐明黄素腺嘌呤二核苷酸（FAD）作为辅因子时，赖氨酸特异性去甲基酶 1（LSD1）催化的组蛋白尾部赖氨酸（H3K4）去甲基化反应中限速胺氧化步骤的催化机制。底物赖氨酸（sLys）的氧化涉及α-CH 键的断裂，同时将一个氢化物离子等价物转移到 FAD，导致亚胺中间体。该氢化物转移途径明显有利于 sLys 氧化，而不是其他提出的机制，包括自由基（或单电子转移）途径以及碳负离子和极性亲核机制。六个 NVT 系综（涵盖不同质子化状态的 sLys 和 K661 以及 K661M 突变体）的 MD 模拟确定了反应 sLys 和 FAD 亚基的两种可能取向（称为“向下”和“向上”）。QM（B3LYP-D/6-31G*）/CHARMM22 水平的计算提供了对机制的分子水平见解，有助于了解 LSD1 如何在无金属环境中激活相当惰性的甲基-CH 键。发现 sLys（向下取向）的适当排列、过渡态稳定化（由于蛋白质环境和有利的轨道相互作用）以及通过加合物形成稳定产物等因素对于促进氧化α-CH 键断裂至关重要。当前的研究还揭示了重要活性位点残基（Y761、K661 和 W695）和保守的水桥基序的作用。Y761 的空间位阻影响有助于正确定位反应伙伴，预测 K661 在底物结合之前去质子化，并作为活性位点碱，从 sLys 接受质子以实现随后的胺氧化，并且由 K661 和 W695 稳定的水桥介导这种质子转移。
