Structure, Mechanism, and Regulation of Quinolinate Synthase, the First Committed Step in Bacterial NAD Biosynthesis

知识优势：烟酰胺腺嘌呤二核苷酸（NAD）是一种必不可少的、普遍存在的代谢物，主要因其在多种生物氧化还原反应中作为共底物而为人所知。然而，最近，它在影响细胞信号传导和染色体重塑的几种非氧化还原反应中所起的作用得到了显著的认可。在细菌中，NAD是由磷酸二羟丙酮和l -天冬氨酸在NadA/NadB酶系统催化下合成的。NadA含有对氧敏感的[4Fe-4S]簇，这是蛋白质功能所必需的；然而，铁硫（Fe/S）簇在催化中的确切作用尚不清楚。此外，蛋白质的活性由二硫醇/二硫化物氧化还原开关调节，其中二硫化物形式的酶比二硫醇形式的活性高约10倍。这种行为很有趣，因为在有氧条件下生长的细菌中NAD及其相关代谢物的浓度明显高于在厌氧条件下生长的细菌。该项目的主要目标是使用x射线晶体学和光谱方法来详细阐明Fe/S簇在催化中的作用。第二个重点领域是阐明伴随可逆二硫化物键形成的结构、化学和电子变化，并确定这些变化如何影响蛋白质的活性和NAD及其相关代谢物的细胞浓度。更广泛的影响：本项目将为培养研究生和本科生严谨、定量的科学方法奠定基础。宾夕法尼亚州立大学在金属酶学方面拥有丰富的专业知识，特别是在研究含铁酶的物理、动力学和计算方法方面。PI将与这里和其他机构的其他教员合作，共同组织一年两次的为期十天的研讨会，培训学生用各种物理方法表征金属蛋白。本项目由分子与细胞生物科学部生物分子动力学、结构与功能研究小组和化学部生命过程化学研究小组共同支持。

Intellectual Merit: Nicotinamide adenine dinucleotide (NAD) is an essential and ubiquitous metabolite known primarily for its role as a co-substrate in a multitude of biological redox reactions. Recently, however, it has received significant recognition for its role in several non-redox reactions that impact cellular signaling and chromosome remodeling. In bacteria, NAD is biosynthesized from dihydroxyacetone phosphate and L-aspartate in a reaction catalyzed by the NadA/NadB enzyme system. NadA contains an oxygen-sensitive [4Fe-4S] cluster, which is required for the protein's function; however, the exact role that the iron-sulfur (Fe/S) cluster plays in catalysis is unclear. Moreover, the activity of the protein is regulated by a dithiol/disulfide redox switch, wherein the disulfide-form of the enzyme is approximately10-fold more active than the dithiol form. This behavior is interesting, because the concentrations of NAD and its associated metabolites are significantly greater in bacteria growing under aerobic conditions than under anaerobic conditions. The major goal of this project is to use X-ray crystallographic and spectroscopic methods to elucidate in detail how the Fe/S cluster functions in catalysis. A second area of emphasis is to elucidate the structural, chemical, and electronic changes that accompany reversible disulfide-bond formation and determine how these changes affect the protein's activity and cellular concentrations of NAD and its related metabolites. Broader Impact: This project will serve as a foundation for training graduate and undergraduate students in rigorous and quantitative scientific methods. Penn State is home to a wealth of expertise in metalloenzymology, especially as it pertains to physical, kinetic, and computational methods for studying iron-containing enzymes. The PI, in collaboration with other faculty here and at other institutions, will co-organize a biannual ten day workshop to train students in various physical methods for the characterization of metalloproteins.This project is jointly supported by the Biomolecular Dynamics, Structure and Function Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes program in the Chemistry Division.