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

细菌 NAD 生物合成的第一步——喹啉酸合酶的结构、机制和调控

• 批准号: 1158486
• 负责人: Squire Booker
• 金额: $ 103万
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1158486&HistoricalAwards=false
• 关键词: Structure; Mechanism; Regulation; Quinolinate; Synthase

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.

智力优点：烟酰胺腺嘌呤二核苷酸 (NAD) 是一种重要且普遍存在的代谢物，主要因其在多种生物氧化还原反应中作为共底物的作用而闻名。然而，最近，它因其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的作用而受到广泛认可。在细菌中，NAD 是在 NadA/NadB 酶系统催化的反应中由磷酸二羟丙酮和 L-天冬氨酸生物合成的。 NadA 含有氧敏感的 [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/二硫键氧化还原开关调节，其中二硫键形式的酶的活性比二硫醇形式高大约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利用 X 射线晶体学和光谱方法详细阐明 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：该项目将作为培训研究生和本科生严格和定量科学方法的基础。宾夕法尼亚州立大学拥有丰富的金属酶学专业知识，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师合作，每年两次共同组织一次为期十天的研讨会，培训学生使用各种物理方法来表征金属蛋白。该项目由分子和细胞生物科学部的生物分子动力学、结构和功能集群以及化学部的生命过程化学项目共同支持。