Lipoic acid biosynthesis: understanding sulfur attachment to aliphatic carbon centers

酶将硫原子附着到惰性碳中心的机制知之甚少，尽管这些反应在细胞代谢和维持中很重要。一种非常重要的此类含硫分子是硫辛酸，其是具有一个附加到碳6（C6）的硫原子和一个附加到碳8（C8）的硫原子的八碳直链脂肪酸（辛酸）。它被用作参与能量产生和储存以及几种氨基酸代谢的几种大型蛋白质复合物的辅因子。尽管对硫辛酸在这些复合物中的功能有相对详细的了解，但由于对硫原子如何附加到脂肪族碳中心的理解有限，因此其生物合成一直令人困惑。本研究旨在评估这一重要含硫分子中附加硫原子的来源及其生物合成机制。 该项目可能为代谢中大范围的C-S键形成过程的这种类型的化学提供新的范例。 开展外展活动，鼓励代表性不足的少数民族学生参与STEM学科。本项目重点关注铁硫簇合物和硫辛酸生物合成的交叉点，旨在提供对脂肪族碳中心如何与硫原子官能化的详细分子理解。实验表明，一种名为NfuA的蛋白质，本身就是一种铁硫蛋白，可以在每次周转后重新插入或修复硫辛酰合酶（利帕）上降解的辅助簇。反过来，NfuA通过复杂的途径获得其簇，通过该途径，铁硫簇被生物合成和运输。本项目的目的是阐明NfuA上的簇如何转移到利帕的详细机制。穆斯堡尔谱和质谱将分别用于跟踪铁和硫的转移。蛋白质晶体学将用于监测硒从含有[4 Fe-4 Se]簇的NfuA转移到利帕，并将努力获得NfuA/利帕复合物的结构。最后，电子顺磁共振光谱，以及蛋白膜伏安法，将被用来研究独特的丝氨酸连接到辅助集群，并确定为什么它是关键的反应利帕。 该项目由分子和细胞生物科学部的分子生物物理学集群和化学部的生命过程化学项目共同资助。

The mechanisms by which enzymes attach sulfur atoms to inert carbon centers are poorly understood despite the importance of these reactions in cellular metabolism and maintenance. One such sulfur-containing molecule of great importance is lipoic acid, an eight-carbon straight-chain fatty acid (octanoic acid) that has one sulfur atom appended to carbon 6 (C6) and one sulfur atom appended to carbon 8 (C8). It is used as a cofactor in several large protein complexes that are involved in energy production and storage, as well as in the metabolism of several amino acids. Despite the relatively detailed understanding of how lipoic acid functions in these complexes, it biosynthesis has been puzzling due to the limited understanding of how sulfur atoms are appended onto aliphatic carbon centers. This research seeks to assess the source of the appended sulfur atoms in this important sulfur-containing molecule and the mechanism of its biosynthesis. This project might provide a new paradigm for this type of chemistry for a large spectrum of C-S bond formation processes in metabolism. Outreach activities to encourage participation of underrepresented minority students in the STEM disciplines will be conducted.This project focuses at the intersection of iron-sulfur cluster and lipoic acid biosynthesis and aims at providing a detailed molecular understanding of how aliphatic carbon centers are functionalized with sulfur atoms. Experiments have shown that a protein called NfuA, which itself is an iron-sulfur protein, can reinsert or repair the degraded auxiliary cluster on lipoyl synthase (LipA) after each turnover. In turn, NfuA obtains its cluster via a complex pathway by which iron-sulfur clusters are biosynthesized and trafficked. The aims of this project focus on elucidating the detailed mechanism that govern how the cluster on NfuA is transferred to LipA. Mössbauer spectroscopy and mass spectrometry will be used to follow iron and sulfur transfer, respectively. Protein crystallography will be used to monitor selenium transfer from NfuA containing a [4Fe-4Se] cluster to LipA, and efforts will be made to obtain structures of the NfuA/LipA complex. Lastly, electron paramagnetic resonance spectroscopy, as well as protein film voltammetry, will be used to study the unique serine ligation to the auxiliary cluster and to determine why it is critical for the reaction of LipA. This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes Program in the Division of Chemistry.