Investigation into the Redox Regulation of the Arabidopsis β‐amylase BAM1

Abstract

Starch in plants is a large polymer of glucose which stores excess glucose for times of limited photosynthesis such as at night. Starch and derivatives of starch are used in many industrial processes such as laundry and papermaking while also playing a nutritional role for humans and other animals. In plants, regulation of starch storage and degradation is a multifaceted system which allows the plant to adjust carbohydrate storage in response to a variety of environmental and stress conditions. However, we have an incomplete picture of how this process is regulated. β-amylases or BAMs hydrolyze starch to produce maltose, a key carbohydrate in plant metabolism. Plants contain many BAM proteins and each has an apparently unique function and regulatory mechanism, although this is an area of active research with many outstanding questions. Sparla and coworkers proposed that BAM1 from the model plant Arabidopsis thaliana, is regulated by the redox state of two amino acids called cysteine which form a crosslink and decrease activity. The molecular basis for this regulation is not clear and is the focus of this project. Small angle X-ray scattering (SAXS) data were collected on reduced BAM1 and the data analyzed in the program RAW to determine solution dimensions of BAM1 in the reduced state. Preliminary analysis of the SAXS data supports the plausibility of the proposed crosslink. The next step within this project is to determine the effect of forming this disulfide bond on the amylase activity. This would be done by activating the disulfide bond within the BAM1 to test the enzymes activity levels through different activity assays. The permanent crosslinks would allow the redox abilities of BAM1 to be tested. Using a fluorescent activity assay for amylases oxidized and reduced BAM1 activity will be compared to correlate our SAXS structures with biochemical function.