Arabidopsis Flavonoid Metabolism as a Model for the Dynamic Enzyme Complex

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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.