b-Glucosidases (EC 3.2.1.21) catalyze the hydrolysis of aryl and alkyl b-D-glucosides as well as b-linked oligosaccharides. In maize, two b-glucosidase isozymes (Glu1 and Glu2) have been characterized with respect to function and structure, including 3-dimensional structure. Certain maize lines were reported not to have b-glucosidase activity (.null.) based on analysis by zymogram techniques. It has been shown that these lines have activity, but the enzyme is not extracted from them because another protein (referred to as BGAF or b-glucosidase aggregating factor hereafter) specifically interacts with Glu1 and Glu2 during extraction, rendering them insoluble. BGAF is related to proteins induced by jasmonic acid (JA), salicylic acid, insect feeding, and salt stress. It is a chimeric protein consisting of two distinct regions: the disease response domain and the jacalin-related lectin (JRL) domain. Like b-glucosidases, BGAF- related proteins are ubiquitous in occurrence. The major goal of this project is to understand b-glucosidase structure-function relationships, how b-glucosidases interact with other proteins such as BGAF and how such interactions affect enzyme function. The specific objectives are: (1) To determine the 3-dimensional structure of intact BGAF and its constituent domains, and study biochemical properties of BGAF-b-glucosidase complexes. (2) To determine the structure and regulation of the genes encoding BGAF in normal and .null. maize lines. (3) To characterize the specific interaction between BGAF and maize b-glucosidases by qualitative and quantitative assays. (4) To identify the site of binding in maize b-glucosidases and BGAF and determine the specific amino acids and their relative importance in b-glucosidase-BGAF interaction using site-directed mutagenesis.
Broader Impact: The results of the proposed research will provide answers to fundamental questions about b-glucosidase and BGAF structure and function and have impact on such areas as cellulosic biomass conversion to glucose, future fuels and products derived from glucose, improved plant productivity and defense without the use of pesticides, and treatment of inherited human syndromes resulting from glycosyl hydrolase defects. Two postdoctoral fellows and two graduate students will be educated during the course of this research. Special effort will be made to recruit these individuals from groups underrepresented in science. In addition, research methods used in this project will form the basis of a laboratory training module for high school teachers and students that will be widely distributed as part of an ongoing educational program.
