STUDIES OF NAD+-DEPENDENT DEACETYLATION AND NAD+ BIOSYNTHETIC ENZYMES

NAD依赖性脱乙酰化和NAD生物合成酶的研究

• 批准号: 7601595
• 负责人: Cynthia Wolberger
• 金额: $ 0.55万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601595
• 关键词: Adenosine Diphosphate Ribose; Binding; Biochemical Reaction; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Deacetylation; Enzymes; Funding; Grant; Hand; Institution; Life; Lysine; Niacinamide; Numbers; Peptides; Reaction; Research; Research Personnel; Resources; Scheme; Sirtuins; Source; Structure; United States National Institutes of Health; enzyme structure; mutant; nicotinamide phosphoribosyltransferase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are requesting beamtime for two related projects in the lab: mechanistic studies of NAD+-dependent deacetylation by sirtuins, and studies of substrate binding to a mammalian NAD+ biosynthetic enzyme. Sir2 enzyme catalyze the NAD+-dependent deacetylation of lysine residues in a two-step mechanism that includes cleavage of nicotinamide from NAD+, followed by transfer of the acetyl group to ADP ribose. While a number of sirtuin structures have been determined, remaining questions center on the precise mechanism of enzymatic reaction. We have devised schemes to trap the reaction O-alkyl amidate intermediate in the crystals and wish to collect data on these crystals. One approach is to use a thio-acetylated peptide, which forms a long-lived covalent complex with ADP ribose. A second approach is to use a mutant enzyme defective in the second step of the reaction, and to soak NAD+ into crystals containing enzyme bound to peptide. Crystals are in hand for all of these. The second project is to continue structural studies of Nampt (nicotinamide phosphoribosyltransferase), whose structure we recently solved in complex with NMN, a product of the reaction. We now wish to determine structures of the enzyme bound to substrate, as well as of the phosphorylated form of the enzyme.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们正在为实验室中的两个相关项目请求 Beamtime：通过 Sirtuins 进行 NAD+ 依赖性脱乙酰化的机制研究，以及与哺乳动物 NAD+ 生物合成酶的底物结合的研究。 Sir2 酶以两步机制催化 NAD+ 依赖性赖氨酸残基脱乙酰化，包括从 NAD+ 上裂解烟酰胺，然后将乙酰基转移到 ADP 核糖。虽然许多沉默调节蛋白的结构已被确定，但剩下的问题集中在酶促反应的精确机制上。我们设计了将反应 O-烷基酰胺中间体捕获在晶体中的方案，并希望收集有关这些晶体的数据。一种方法是使用硫代乙酰化肽，它与 ADP 核糖形成长寿命的共价复合物。第二种方法是使用反应第二步中存在缺陷的突变酶，并将 NAD+ 浸入含有与肽结合的酶的晶体中。所有这些，水晶都在手。第二个项目是继续研究 Nampt（烟酰胺磷酸核糖基转移酶）的结构，我们最近解析了其与反应产物 NMN 的复合物结构。我们现在希望确定与底物结合的酶的结构，以及酶的磷酸化形式。