Crystallographic Analysis of NMN Adenylytransferases

NMN 腺苷酸转移酶的晶体学分析

• 批准号: 6459370
• 负责人: HONG ZHANG
• 金额: $ 21.9万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-15 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6459370
• 关键词: DNA binding protein; Escherichia coli; Haemophilus influenzae; Salmonella typhimurium; X ray crystallography; adenosine triphosphate; bacterial proteins; crystallization; dinucleotide; enzyme mechanism; enzyme substrate complex; gene expression; nicotinamide; nicotinamide adenine dinucleotide; nicotinamide ribotide; nucleotidyltransferase; oligonucleotides; polymerase chain reaction; protein purification; protein structure function; site directed mutagenesis

DESCRIPTION: (provided by applicant) Nicotinamide mononucleotide adenylyltransferase (NMNAT) is an indispensable enzyme in the biosynthesis and salvage of NAD and NADP in all living organisms. In prokaryotes, it is absolutely required for cell survival, thus representing an attractive target for designing new broad-spectrum anti-infectious pharmaceutics. There is also considerable medical interest in human NMNAT because it catalyzes the key step in the metabolic conversion of the antitumor drug tiazofurin to its active form, tiazofurin adenine dinucleotide (TAD). Because of the vital roles of NAD in the cell, adequate levels of NAD must be maintained. In bacteria, a single protein NadR performs multiple functions in response to the cellular NAD and ATP levels. These include a transcriptional repressor, a NMN adenlyltransferase, and a nicotinamide ribose kinase. NadR regulates the expression of the genes involved in both de novo biosynthesis and savage of NAD. It also controls the uptake of NMN and its subsequent adenylation. Structural information of NMNAT and NadR, along with their complexes with substrates will be essential for understanding the catalysis, substrate recognition, inhibition, as well as regulation of these important enzymes. We have solved the crystal structure of the first bacterial NMNAT in its ligand free form and have obtained well diffracting human NMNAT crystals. Comparison of the bacterial and human enzyme structures will help to design specific bacterial inhibitors with high selectivity. Additionally, we have expressed and purified NadR proteins from S. typhimurium and H. influenzae, and the crystallization trials are in progress. Elucidating the structures of NadR in its various ligand-bound forms and its complex with DNA will reveal how its conformation and function are modulated by both NAD and ATP.

描述：（申请人提供）烟酰胺单核苷酸 腺苷酸转移酶（NMNAT）是生物合成和代谢过程中不可缺少的酶。 挽救所有生物体中的 NAD 和 NADP。在原核生物中，它是 细胞生存绝对必需的，因此是一个有吸引力的目标 用于设计新的广谱抗感染药物。还有 人们对人类 NMNAT 具有相当大的医学兴趣，因为它催化了关键步骤 抗肿瘤药物噻唑呋林代谢转化为其活性物质 形式，噻唑呋林腺嘌呤二核苷酸（TAD）。由于 NAD 的重要作用 在细胞中，必须维持足够的 NAD 水平。在细菌中，单个 蛋白质 NadR 响应细胞 NAD 执行多种功能， ATP 水平。其中包括转录抑制因子、NMN 腺苷酸转移酶和烟酰胺核糖激酶。 NadR 调节 参与从头生物合成和野蛮的基因的表达 辅酶A。它还控制 NMN 的摄取及其随后的腺苷酸化。 NMNAT 和 NadR 的结构信息，以及它们的复合物 底物对于理解催化作用至关重要 这些重要酶的识别、抑制和调节。我们 解析了第一个细菌 NMNAT 配体的晶体结构 自由形式并获得了良好衍射的人类 NMNAT 晶体。比较 细菌和人类酶结构的研究将有助于设计特定的 具有高选择性的细菌抑制剂。此外，我们已经表达并 从鼠伤寒沙门氏菌和流感嗜血菌中纯化的 NadR 蛋白，以及 结晶试验正在进行中。阐明 NadR 的结构 它的各种配体结合形式及其与 DNA 的复合物将揭示其如何 构象和功能由 NAD 和 ATP 共同调节。