Crystallographic Analysis of NMN Adenylytransferases

NMN 腺苷酸转移酶的晶体学分析

• 批准号: 6752498
• 负责人: HONG ZHANG
• 金额: $ 19.5万
• 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/6752498
• 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.

性状：（由申请人提供）烟酰胺单甘肽 腺苷酰转移酶（NMNAT）是生物合成中不可缺少的酶， 挽救所有生物体中的NAD和NADP。在原核生物中， 这是细胞生存所必需的，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 人类NMNAT具有相当大的医学兴趣，因为它催化关键步骤 在抗肿瘤药物硫唑弗林代谢转化为其活性 形式，噻唑弗林腺嘌呤二核苷酸（tiazofurin adenine dinucleotide，IATA）。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， 蛋白质NadR响应于细胞NAD执行多种功能， ATP水平。这些包括转录抑制因子，NMN 腺苷酰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， NAD。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，沿着它们与 底物对于理解催化作用是必不可少的， 识别、抑制以及调节这些重要的酶。我们 已经解决了第一个细菌NMNAT在其配体中的晶体结构 游离形式，并获得了衍射良好的人NMNAT晶体。比较 细菌和人类酶的结构将有助于设计特定的 具有高选择性的细菌抑制剂。此外，我们还表示， 从S.鼠伤寒沙门氏菌和H.流感，以及 结晶试验正在进行中。阐明了NadR在 它的各种配体结合形式及其与DNA的复合物将揭示它是如何与DNA结合的。 构象和功能由NAD和ATP调节。