Subcellular Organization of the Flavonoid Enzyme Complex

类黄酮酶复合物的亚细胞组织

• 批准号: 0131010
• 负责人: Brenda Winkel
• 金额: $ 38.21万
• 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: 2002
• 资助国家: 美国
• 起止时间: 2002-03-15 至 2006-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0131010&HistoricalAwards=false
• 关键词: Subcellular; Organization; Flavonoid; Enzyme; Complex

Enzymes function within cells to catalyze biochemical reactions, thereby controlling the amounts and types of products that are produced by different types of cells under different conditions. Work in many laboratories has provided evidence that enzymes that cooperate in biosynthetic pathways and other coordinated systems, such as DNA replication and protein synthesis, are often physically associated as macromolecular complexes. This organization has the potential to dramatically enhance the biochemical efficiency of living cells as well as providing mechanisms for sequestering toxic or volatile intermediates, regulating competition among branch pathways, and coordinating interdependent processes. Some of these systems, such as the machinery of protein and nucleic acid biosynthesis, are extremely stable and can be extracted from cells as intact multienzyme structures. Others, such as the TCA cycle and the glycolytic pathway, are organized as "dynamic" complexes that may dissociate and reform in response to environmental or physiological stimuli. However, there are only a few cases in which a specific physiological function for this organization has been documented. At the same time, very little is known about the molecular basis of enzyme complex formation and localization. This project aims to address these questions using the flavonoid biosynthetic pathway in the plant, Arabidopsis, as an experimental system. Numerous genetic, molecular, and biochemical tools are available for this system, including cloned genes for seven major flavonoid enzymes, purified recombinant enzymes produced in bacterial cells, polyclonal antibodies against many of these proteins, and a collection of mutants that includes null alleles for several key enzymatic steps. The major goals of the current project are to 1) determine the three-dimensional structures of two of the flavonoid enzymes, 2) use new technologies to study the interactions between these enzymes, 3) track changes in the subcellular locations of the enzymes in response to environmental stimuli such as wounding and gravity, and 4) study the biochemical and physiological effects of targeting flavonoid enzymes to inappropriate locations within the plant cell. Expanding the knowledge of the structure and function of enzyme complexes is essential to developing a complete understanding of how cells organize and regulate metabolic activity. This information is crucial for efforts to alter cellular metabolism via gene therapy and in the development of transgenic organisms for industrial and agricultural applications. This project will also provide a training ground in contemporary molecular genetic and biochemical technologies for undergraduate and graduate students.

酶在细胞内起催化生化反应的作用，从而控制不同类型的细胞在不同条件下产生的产物的量和类型。 许多实验室的工作提供了证据，证明在生物合成途径和其他协调系统（如DNA复制和蛋白质合成）中合作的酶通常以大分子复合物的形式物理结合。 这种组织有可能显著提高活细胞的生化效率，并提供隔离有毒或挥发性中间体的机制，调节分支途径之间的竞争，并协调相互依赖的过程。 其中一些系统，如蛋白质和核酸生物合成的机制，是非常稳定的，可以从细胞中提取完整的多酶结构。 其他的，如TCA循环和糖酵解途径，被组织为“动态”复合物，其可以响应于环境或生理刺激而解离和改革。 然而，只有少数情况下，该组织的特定生理功能已被记录在案。 同时，对酶复合物形成和定位的分子基础知之甚少。 本项目旨在解决这些问题，使用植物中的类黄酮生物合成途径，拟南芥，作为一个实验系统。 许多遗传，分子和生物化学工具可用于该系统，包括7种主要类黄酮酶的克隆基因，细菌细胞中产生的纯化重组酶，针对许多这些蛋白质的多克隆抗体，以及包括几个关键酶促步骤的无效等位基因的突变体的集合。 目前项目的主要目标是：1）确定两种类黄酮酶的三维结构，2）使用新技术研究这些酶之间的相互作用，3）跟踪酶的亚细胞位置对环境刺激（如创伤和重力）的反应变化，以及4）研究将类黄酮酶靶向到植物细胞内的不适当位置的生物化学和生理学效应。 扩展酶复合物的结构和功能的知识对于全面了解细胞如何组织和调节代谢活动至关重要。 这一信息对于通过基因治疗改变细胞代谢的努力以及工业和农业应用的转基因生物的开发至关重要。 该项目还将为本科生和研究生提供现代分子遗传和生物化学技术的培训基地。