Subcellular Organization of the Flavonoid Enzyme Complex

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数量和种类。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存在物理关联。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，调节分支通路之间的竞争，协调相互依存的过程。其中一些系统，如蛋白质和核酸生物合成机制，是非常稳定的，可以作为完整的多酶结构从细胞中提取出来。其他的，如三羧酸循环和糖酵解途径，被组织为“动态”复合物，可以在环境或生理刺激下解离和重组。然而，只有少数情况下，该组织的特定生理功能已被记录。同时，对酶复合体形成和定位的分子基础了解甚少。本项目旨在利用拟南芥中的类黄酮生物合成途径作为实验系统来解决这些问题。许多遗传、分子和生化工具可用于该系统，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，以及包含几个关键酶促步骤的零等位基因的突变体集合。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)利用新技术研究这些酶之间的相互作用；3)跟踪酶在响应环境刺激（如伤害和重力）时亚细胞位置的变化；4)研究将类黄酮酶靶向到植物细胞内不适当位置的生化和生理效应。扩大对酶复合物结构和功能的了解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用于工业和农业应用的转基因生物的发展至关重要。该项目还将为本科生和研究生提供当代分子遗传和生化技术的培训基地。

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.