Arabidopsis Flavonoid Metabolism as a Model for the Dynamic Enzyme Complex

拟南芥类黄酮代谢作为动态酶复合物的模型

• 批准号: 9808117
• 负责人: Brenda Winkel
• 金额: $ 30万
• 依托单位: Virginia Polytechnic Institute and State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808117&HistoricalAwards=false
• 关键词: Arabidopsis; Flavonoid; Metabolism; Model; Dynamic

Shirley The assembly of cooperating enzymes into stable complexes is a well-established feature of cell metabolism. This organization provides the means to attain high local substrate concentrations, regulate competition among branch pathways, coordinate the activities of interdependent pathways, and sequester toxic or volatile intermediates from the rest of the cell. Unfortunately, many of these complexes are relatively shortlived or unstable and are therefore notoriously difficult to study using conventional biochemical and kinetic approaches. However, powerful new technologies are now emerging that offer promise for identifying the molecular mechanisms underlying the assembly of dynamic enzyme complexes and uncovering the true biological significance of these macromolecular structures. The flavonoid biosynthetic pathway of Arabidopsis provides a particularly useful experimental model for studying enzyme complex assembly and regulation. Numerous genetic, molecular and biochemical tools have been developed for this system, including cloned genes for seven major flavonoid enzymes,purified recombinant enzymes produced in bacterial cells, polyclonal antibodies against five of these proteins, and a collection of mutants that includes null alleles for several key enzymatic steps. Direct evidence has recently been obtained for interaction among the first several enzymes of the fiavonoid pathway using two-hybrid, affinity chromatography, and co-immunolocalization analyses. The major goals of the project are to define the protein domains that mediate interactions among these enzymes and to characterize the subcellular organization of the flavonoid enzyme complex. The project will also develop methods for the future analysis of the assembly of this complex in response to specific physiological cues. Protein interaction domains will be identified in vitro by extending the results of existing affinity chromatography assays. Interactions between full-length and truncated flavonoid enzymes fused to green fluorescent protein (GFP) will be tested in fluorescence resonance energy transfer (FRET) assays in bacterial cells. Experiments will also be aimed at examining interactions in transgenic plants, both by expressing phage-derived antibodies directed against specific interacting domains and using GFP-FRET. Immunolocalization, together with fluorescence and confocal laser microscopy of GFP fusion proteins, will be used to study the subcellular organization of the flavonoid enzyme complex and to begin to track its assembly in living cells. This project aims to improve our understanding of the organization of biosynthetic pathways within cells and the importance of this organization for the control of metabolism. Specifically, the project will determine the molecular details underlying assembly of the Arabidopsis fiavonoid pathway into an enzyme complex. The work will establish the basis for future efforts to study the biological significance of controlled complex assembly in response to developmental and environmental cues. It will also provide information essential to efforts aimed at introducing new catalytic activities into existing metabolic pathways. A detailed understanding of the structure and regulation of the flavonoid enzyme system will contribute information essential to the rational engineering of metabolism in living systems, with applications ranging from agriculture to human gene therapy.

协同作用的酶组装成稳定的复合物是细胞代谢的一个公认特征。这种组织提供了获得高局部底物浓度的手段，调节分支途径之间的竞争，协调相互依赖的途径的活动，并从细胞的其余部分隔离有毒或挥发性中间体。不幸的是，许多这些复合物的寿命相对较短或不稳定，因此很难使用传统的生化和动力学方法进行研究。然而，强大的新技术正在出现，为确定动态酶复合物组装的分子机制和揭示这些大分子结构的真正生物学意义提供了希望。拟南芥类黄酮生物合成途径为研究酶复合物的组装和调控提供了一个特别有用的实验模型。许多遗传，分子和生物化学的工具已经开发了这个系统，包括克隆基因的七个主要的类黄酮酶，纯化的重组酶产生的细菌细胞，多克隆抗体对这些蛋白质中的五个，和一个集合的突变体，包括无效等位基因的几个关键酶的步骤。最近已经获得了直接的证据之间的相互作用的前几个酶的fiavlastine途径，使用双杂交，亲和层析，共免疫定位分析。该项目的主要目标是确定介导这些酶之间相互作用的蛋白质结构域，并表征类黄酮酶复合物的亚细胞组织。该项目还将开发用于未来分析这种复合物响应特定生理线索的组装的方法。蛋白质相互作用域将通过扩展现有亲和层析测定的结果在体外鉴定。融合到绿色荧光蛋白（GFP）的全长和截短的类黄酮酶之间的相互作用将在细菌细胞中的荧光共振能量转移（FRET）测定中进行测试。实验也将旨在研究转基因植物中的相互作用，通过表达针对特定相互作用结构域的噬菌体衍生抗体和使用GFP-FRET。免疫定位，连同荧光和共聚焦激光显微镜的GFP融合蛋白，将用于研究的类黄酮酶复合物的亚细胞组织，并开始跟踪其在活细胞中的组装。 该项目旨在提高我们对细胞内生物合成途径的组织以及这种组织对代谢控制的重要性的理解。具体来说，该项目将确定拟南芥fiaviruspathway组装成酶复合物的分子细节。这项工作将为未来研究控制复杂组装对发育和环境线索的生物学意义奠定基础。它还将为旨在将新的催化活性引入现有代谢途径的努力提供必要的信息。对类黄酮酶系统的结构和调节的详细了解将为生命系统中代谢的合理工程提供必要的信息，其应用范围从农业到人类基因治疗。