Characterization of the Allosteric Regulation of BAM2 in Arabidopsis thaliana

Abstract

Starch is an important component of the human diet as it is broken down into glucose to provide energy for metabolic reactions in the body. The amount and sources of starch that humans consume greatly varies around the world depending on the plants that are indigenous to each diet. In plants, starch is hydrolyzed into the disaccharide maltose by β‐amylase enzymes, a process that is important night time metabolism and the response to stress in plants. By understanding starch regulation by β‐amylases, we could potentially improve the quality of food crops which will benefit those who suffer from food insecurity and nutritional deficiencies. Arabidopsis thaliana contains nine β‐amylases (BAMs) that show distinct catalytic activities and functional regulation. Starch is located in the chloroplast and there are 4 chloroplastic BAMs. We are interested in understanding the functional and regulatory differences in these enzymes. The focus of our study is BAM2, which was recently found to be a tetramer and contains a secondary starch binding site which regulates BAM2 activity. Since there are no structures of BAMs from Arabidopsis, we aimed to characterize the structural mechanism of BAM2 regulation by starch binding to the secondary binding site. We first modeled BAM2 as a tetramer using previous experimental information and then performed simulations of this model in several substrate and starch bound states to understand the role of the secondary starch binding site on the active structure. We found that starch binding influences protein dynamics near the active site, which we suggest may be the basis for allosteric regulation. We further identified an amino acid which appears to regulate the T to R transition of the enzyme. We supported these computational studies with small angle X‐ray scattering studies of BAM2 structure and with biochemical assays of amylase activity. These latter studies were aided by the development of a novel fluorescent biosensor assay of maltose production that allows for comparison of diverse substrates from small polysaccharides to soluble starch within a single assay method. These comparisons indicate that BAM2 strongly prefers starch‐like substrates over shorter polysaccharides, likely due to allosteric regulation by the secondary starch binding site. Current studies are focused on understanding the effects of this molecular mechanism on starch structure with the goal of elucidating the functional role of BAM2 in plants.Support or Funding InformationNSF‐RUI MCB‐1616467NSF REU CHE‐17578744‐VA OrganizationJeffrey E. Tickle ScholarshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.