Lipoic acid biosynthesis: understanding sulfur attachment to aliphatic carbon centers

尽管这些反应在细胞新陈代谢和维持中很重要，但酶将硫原子连接到惰性碳中心的机制却知之甚少。硫辛酸是一种非常重要的含硫分子，它是一种八碳直链脂肪酸(辛酸)，其碳6(C6)上有一个硫原子，碳8(C8)上有一个硫原子。它被用作几个大型蛋白质复合体中的辅因子，这些复合体参与能量的产生和储存，以及几种氨基酸的新陈代谢。尽管对硫辛酸在这些络合物中的功能有相对详细的了解，但由于对硫原子如何连接到脂肪族碳中心的了解有限，硫辛酸的生物合成一直令人困惑。本研究旨在评估这一重要的含硫分子中加成硫原子的来源及其生物合成机理。这个项目可能会为这类化学提供一种新的范式，用于研究代谢过程中大量的C-S键的形成过程。将开展推广活动，鼓励未被充分代表的少数族裔学生参与STEM学科。本项目侧重于铁-硫簇和硫辛酸生物合成的交叉点，旨在提供关于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的蛋白质本身是一种铁硫蛋白质，可以在每次更替后重新插入或修复硫磺合成酶(LIPA)上降解的辅助簇。反过来，NfuA通过一种复杂的途径获得其簇，通过该途径铁-硫簇被生物合成和交易。该项目的目的是阐明管理NfuA上的集群如何转移到LIPA的详细机制。穆斯堡尔光谱和质谱学将分别用于跟踪铁和硫的转移。蛋白质结晶学将被用来监测硒从含有[4Fe-4Se]簇的NfuA向LIPA的转移，并努力获得NfuA/LIPA络合物的结构。最后，电子顺磁共振波谱，以及蛋白质膜伏安法，将被用来研究独特的丝氨酸与辅助簇的连接，并确定为什么它对LIPA的反应至关重要。该项目由分子和细胞生物科学部的分子生物物理组和化学部的生命过程化学计划共同资助。

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.