STRUCTURAL DETERMINATIONS OF GLUTAMINE-DEPENDENT NAD SYNTHETASES

谷氨酰胺依赖性 NAD 合成酶的结构测定

• 批准号: 8363346
• 负责人: Barbara Gerratana
• 金额: $ 0.19万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363346
• 关键词: Active Sites; Ammonia; Anabolism; Binding; Cell Line; Citrates; Complex; Crystallization; Crystallography; Disease; Drug Delivery Systems; Enzyme Tests; Enzymes; Funding; Glutaminase; Glutamine; Grant; Human; Insecta; Ions; Ligands; Ligase; Light; Mediating; Modeling; Molecular; Molecular Conformation; Mycobacterium tuberculosis; National Center for Research Resources; Pathway interactions; Principal Investigator; Recycling; Regulation; Research; Research Infrastructure; Resolution; Resources; Sequence Homology; Shapes; Source; Structure; Synchrotrons; United States National Institutes of Health; Work; ammonium citrate; analog; cost; design; inhibitor/antagonist; retinal rods

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Glutaminedependent NAD+ synthetase catalyzes the last step of the de novo and recycling pathways in NAD+ biosynthesis. This enzyme is essential for survival of replicating and non-replicating Mycobacterium tuberculosis and as such it is an attractive drug target. In humans NAD+ synthetase independent pathways participate in NAD+ biosynthesis. We are elucidating the mechanism of regulation of the catalytic activities at active sites that are spatially separated, determining structural and mechanistic differences between the H. sapiens and M. tuberculosis enzymes and testing both kinetically and structurally inhibitors, resulting in the design of inhibitors specific for NAD+ synthetaseTB. The structure of wild type NAD+ synthetaseTB in complexes with DON and NaAD, previously solved in our lab, is a homoctameric and features a 40 ¿ long inter-subunit ammomia tunnel for transport of ammonia from glutaminase active site to the synthetase active site. We have been solved four structures of NAD+ synthetaseTB with different ligand bounds at range of 2.6-3.0 ¿ resolutions. In our attempt to obtain a structure in complexes with ligands that stabilized active site loops P2 at the synthetase active site. We hypothesize that ordering of loop P2 induces a conformational change that activates the glutaminase active site. The crystals were co-crystallized in 1.4-1.8 M ammonium citrate as wild type does. The structure complexes showed the binding of ATP or AMP and NaAD at the synthetase active site but Mg2+ or PPi/Mg2+ were not found. Mg2+ ions were found to stabilize loop P2 by coordinating with ATP or NaAD-AMP intermediate and the use of high ammonia citrate at high concentration as a precipitant may interfere with the coordination mediated by Mg2+ ions, resulting in loop P2 were still disordered in any structures solved. Now, we have several crystals with analog of NaAD-AMP and PPi at the synthetase active site (10 rod-shaped, 2 plate-like, and 4 pyramid-shaped crystals), which were crystallized in three brand new crystallization conditions. These crystals may provide the different space group with structural information of loop P2 and the activated conformation. In addition, we are currently working on the human enzyme, which we can get expressed in Sf9 insect cell line. Crystals of native enzyme with analog of NaAD-AMP and PPi were collected at the SSRL syntchrostron at 3.0 ¿ resolution and we are solving structure using molecular replacement. If it fails due to the low sequence homology of the human NAD synthetase with search model we will need to generate heavy-atom derivatives and solve the structure by SAD/MAD.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 谷氨依赖性NAD+合成酶催化NAD+生物合成中从头和再循环途径的最后一步。这种酶对于复制型和非复制型结核分枝杆菌的存活是必需的，因此它是一种有吸引力的药物靶标。在人类中，NAD+合成酶非依赖性途径参与NAD+生物合成。我们正在阐明在空间上分离的活性位点的催化活性的调节机制，确定H。sapiens和M.结核病酶和测试动力学和结构抑制剂，导致特异性NAD+合成酶TB的抑制剂的设计。野生型NAD+合成酶TB与DON和NaAD的复合物的结构，以前在我们的实验室中解决，是一个同八聚体，并具有40 ½长的亚基间氨通道，用于将氨从转氨酶活性位点转运到合成酶活性位点。我们已经解决了四个结构的NAD+ synthetaseTB与不同的配体结合在2.6-3.0 <$分辨率范围。在我们试图获得与稳定活性位点环P2在合成酶活性位点的配体的复合物的结构。我们推测，环P2的顺序诱导构象变化，激活转氨酶活性位点。晶体在1.4- 1.8M柠檬酸铵中共结晶，与野生型一样。结果表明，在合成酶活性位点上，ATP或AMP与NaAD结合，但未发现Mg ~（2+）或PPi/Mg ~（2+），Mg ~（2+）通过与ATP或NaAD-AMP中间体配位而稳定环P2，高浓度柠檬酸铵沉淀可能干扰Mg ~（2+）介导的配位作用，导致环P2在任何解出的结构中仍然是无序的。现在，我们有几种在合成酶活性位点具有NaAD-AMP和PPi类似物的晶体（10个棒状，2个板状和4个棒状晶体），它们在三种全新的结晶条件下结晶。这些晶体可以提供不同空间群的环P2和活化构象的结构信息。此外，我们目前正在研究人类酶，我们可以在Sf 9昆虫细胞系中表达。在SSRL同步加速器中以3.0？分辨率收集具有NaAD-AMP和PPi类似物的天然酶晶体，我们正在使用分子置换来解析结构。如果搜索模型由于人NAD合成酶的低序列同源性而失败，则需要生成重原子衍生物并通过SAD/MAD求解结构。