Mechanism of Interaction Between Maize Beta-Glucosidases and a Chimeric Protein Containing Disease Response and Lectin Domains

β-葡糖苷酶（EC www.example.com）催化芳基和烷基β-D-葡糖苷以及β-连接寡糖的水解。 在玉米中，两种β-葡萄糖苷酶同工酶（Glu 1和Glu 2）的特征在于功能和结构，包括三维结构。 据报道，某些玉米品系不具有β-葡糖苷酶活性（.基于酶谱技术的分析。 已经表明，这些细胞系具有活性，但是酶没有从它们中提取出来，因为另一种蛋白质（以下称为BGAF或β-葡糖苷酶聚集因子）在提取过程中特异性地与Glu 1和Glu 2相互作用，使它们不溶。 BGAF与茉莉酸（JA）、水杨酸、昆虫取食和盐胁迫诱导的蛋白有关。 它是一种嵌合蛋白，由两个不同的区域组成：疾病反应结构域和黑素相关凝集素（JRL）结构域。 与β-葡萄糖苷酶一样，BGAF相关蛋白普遍存在。 该项目的主要目标是了解b-葡萄糖苷酶的结构-功能关系，b-葡萄糖苷酶如何与其他蛋白质如BGAF相互作用，以及这种相互作用如何影响酶的功能。 具体目标是：（1）确定完整BGAF及其组成结构域的三维结构，并研究BGAF-β-葡萄糖苷酶复合物的生化性质。 (2)确定正常人和非正常人BGAF编码基因的结构和调控。玉米品系 (3)通过定性和定量分析来表征BGAF与玉米β-葡萄糖苷酶之间的特异性相互作用。 (4)鉴定玉米β-葡萄糖苷酶和BGAF的结合位点，并使用定点突变确定β-葡萄糖苷酶-BGAF相互作用中的特定氨基酸及其相对重要性。 更广泛的影响：拟议研究的结果将为有关b-葡萄糖苷酶和BGAF结构和功能的基本问题提供答案，并对纤维素生物质转化为葡萄糖，未来燃料和葡萄糖衍生产品，提高植物生产力和防御等领域产生影响不使用农药，以及治疗由糖基水解酶缺陷引起的遗传性人类综合征。 两名博士后研究员和两名研究生将在本研究过程中接受教育。 将特别努力从科学界代表性不足的群体中招募这些人。 此外，本项目中使用的研究方法将成为高中教师和学生实验室培训模块的基础，该模块将作为正在进行的教育计划的一部分广泛分发。

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.