Department of Chemistry, Faculty of Science and Letters, Istanbul Technical University, Maslak, Istanbul 34469, Turkey.
J Phys Chem A. 2021 Nov 4;125(43):9459-9477. doi: 10.1021/acs.jpca.1c05397. Epub 2021 Oct 22.
Recently, two new mechanistic proposals for the kynurenine 3-monooxygenase (KMO) catalyzed hydroxylation reaction of l-Kynurenine (l-Kyn) have been proposed. According to the first proposal, instead of the distal oxygen, the proximal oxygen of the hydroperoxide intermediate of flavin adenine dinucleotide (FAD) is transferred to the substrate ring. The second study proposes that l-Kyn participates in its base form in the reaction. To address these proposals, the reaction was reconsidered with a 386 atom quantum cluster model that is based on a recent X-ray structure (PDB id: 6FOX). The computations were carried out at the UB3LYP/6-311+G(2d,2p)//UB3LYP/6-31G(d,p) level with solvation (polarizable continuum model) and dispersion (DFT-D3(BJ)) corrections. To supplement the results of the density functional theory (DFT) calculations, molecular dynamics (MD) simulations of the protein-substrate complex were employed. The comparison of a proximal oxygen transfer mechanism to the distal oxygen transfer mechanism revealed that the former requires too high of a barrier energy while the latter validated our previous results. According to the MD simulations, the hydroperoxy moiety does not favor an alignment that might promote the proximal oxygen transfer mechanism. In the second part of the study, hydroxylation reaction with the base form of l-Kyn was sought. Although DFT calculations confirmed a much more facile reaction with the base form of l-Kyn, a mechanism which would allow the deprotonation of the l-Kyn before the oxygen transfer could not be determined with the X-ray-based positions. A concerted mechanism with both the oxygen transfer and the deprotonation required a high barrier energy. A stepwise mechanism involving the deprotonation of l-Kyn was found, starting from an MD frame. The overall barrier of the oxygen transfer step of this model was found to be in the range of that of with neutral l-Kyn. MD simulations supported the idea of ineffectiveness of the nearby shell surrounding the utilized active site core on the deprotonation of l-Kyn.
最近，提出了两种关于犬尿氨酸 3-单加氧酶（KMO）催化 l-犬尿氨酸（l-Kyn）羟化反应的新的机制建议。根据第一个建议，不是远端氧，而是黄素腺嘌呤二核苷酸（FAD）的过氧化物中间体的近端氧被转移到底物环上。第二项研究提出，l-Kyn 在反应中以其碱基形式参与。为了解决这些建议，反应在基于最近的 X 射线结构（PDB id：6FOX）的 386 个原子量子簇模型上重新进行了考虑。计算是在 UB3LYP/6-311+G(2d,2p)//UB3LYP/6-31G(d,p)水平上进行的，带有溶剂化（极化连续体模型）和色散（DFT-D3(BJ)）修正。为了补充密度泛函理论（DFT）计算的结果，还进行了蛋白质-底物复合物的分子动力学（MD）模拟。与远端氧转移机制相比，近端氧转移机制的比较表明，前者需要过高的势垒能量，而后者验证了我们之前的结果。根据 MD 模拟，过氧基团不利于可能促进近端氧转移机制的对齐。在研究的第二部分，寻求 l-Kyn 碱基形式的羟化反应。尽管 DFT 计算证实了与 l-Kyn 碱基形式的反应更容易，但不能根据基于 X 射线的位置确定允许 l-Kyn 去质子化之前发生氧转移的机制。需要高势垒能量的协同机制同时发生氧转移和去质子化。从 MD 帧开始发现了涉及 l-Kyn 去质子化的逐步机制。该模型的氧转移步骤的总势垒被发现处于与中性 l-Kyn 相同的范围内。MD 模拟支持了利用活性位点核心周围的近壳对 l-Kyn 去质子化无效的想法。
