Determining the Functional and Structural Domains of Arabidopsis Flavonoid Enzymes

Biosynthetic pathways are often organized within cells as groups of interacting enzymes or enzyme complexes. Although this organization appears to be widespread in biological systems, it is still poorly understood. The flavonoid pathway of Arabidopsis thaliana has been developed in our laboratory as a model for studying enzyme complexes. Several of the enzymes of this pathway have been shown to interact in vitro, and have also been found to be co-localized in roots. Our studies are currently focusing on chalcone synthase (CHS) and chalcone isomerase (CHI), which catalyze the first two committed steps in flavonoid biosynthesis, and how they interact with each other to form a multi-enzyme complex. In our effort to better understand the dynamics of this multi-enzyme complex, we are utilizing small angle neutron scattering as a tool to determine the stoichiometry and physical locations of these interactions. Data collected from the SANS experiments are now being used to build a model of CHS and CHI together in complex, which hopefully can be used to predict the interfaces involved in the CHS-CHI interaction, which can then be further tested by site directed mutagenesis. These experiments should provide new insights into the self-assembly of enzyme complexes.

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