Subcellular Organization of the Flavonoid Enzyme Complex

Virginia Polytechnic Institute And State University, Blacksburg VA

Investigators

Abstract

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.