Molecular Simulations and Design Group, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany.
Org Biomol Chem. 2024 Aug 14;22(32):6550-6560. doi: 10.1039/d4ob00862f.
Kynurenine 3-monooxygenase (KMO) regulates the levels of important physiological intermediates in the kynurenine pathway [Guillemin, , , 2007, , 12884], which is the major route for L-tryptophan catabolism. Its catalytic activity (hydroxylation) is dependent on the formation of a short-lived intermediate that forms after the reduction of the coenzyme FAD. The reduction takes place fast when the substrate binds to KMO. Crystal structures of the apo form and in complex with an effector inhibitor, which prevents the hydroxylation of the substrate but also stimulates KMO like the substrate, and a competitive inhibitor, which suppresses the substrate hydroxylation, are available for the resting conformation only. The active conformational state that enables the reduction of FAD at an exposed location of KMO after its stimulation by an effector, however, was implicated but not resolved experimentally and has remained elusive so far. Molecular dynamics simulations of apo KMO and the inhibitor-KMO complexes are carried out using extensive multi-dimensional umbrella sampling to explore the free-energy surface of the coenzyme FAD's conformational conversion from the state (buried within the active site) to the state. This allows a discussion and comparison with the experimental results, which showed a significant increase in the rate of reduction of FAD in the presence of an effector inhibitor and absence of enzymatic function in the presence of a competitive inhibitor [Kim, , , 2018, , 426]. The free-energy barriers associated with those conformational changes and structural models for the active conformation are obtained. The interactions during the conformational changes are determined to identify the influence of the effector.
犬尿氨酸 3-单加氧酶（KMO）调节犬尿氨酸途径中重要生理中间产物的水平[Guillemin 等人，2007 年，12884]，该途径是 L-色氨酸分解代谢的主要途径。其催化活性（羟化）依赖于辅酶 FAD 还原后形成的短暂中间产物。当底物与 KMO 结合时，还原过程很快发生。apo 形式和与效应物抑制剂复合物的晶体结构，该抑制剂可阻止底物的羟化，但也像底物一样刺激 KMO，以及竞争性抑制剂，可抑制底物的羟化，仅适用于静止构象。然而，能够在效应物刺激后使 FAD 在 KMO 的暴露位置还原的活性构象状态是暗示的，但尚未通过实验解决，迄今为止仍然难以捉摸。使用广泛的多维伞状采样对 apo KMO 和抑制剂-KMO 复合物进行分子动力学模拟，以探索辅酶 FAD 构象从 态（埋藏在活性部位内）到 态的构象转换的自由能表面。这允许与实验结果进行讨论和比较，实验结果表明，在存在效应物抑制剂的情况下，FAD 的还原速率显著增加，而在存在竞争性抑制剂的情况下则缺乏酶功能[Kim 等人，2018 年，426]。获得了与这些构象变化相关的自由能障碍和活性构象的结构模型。确定构象变化期间的相互作用以识别效应物的影响。
