The Mechanism of Substrate (aglycone) Specificity and Binding in Beta-Glucosidases

β-葡萄糖苷酶的底物（糖苷配基）特异性和结合机制

• 批准号: 9905698
• 负责人: Asim Esen
• 金额: $ 36万
• 依托单位: Virginia Polytechnic Institute and State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-10-01 至 2004-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9905698&HistoricalAwards=false
• 关键词: Mechanism; Substrate; aglycone; Specificity; Binding

9905698EsenThe major goal of this research is to understand the mechanism of substrate (i.e., aglycone) specificity in the enzyme Beta-glucosidase (Beta-D-glucoside glucohydrolase, EC 3.2.1.21). Maize and sorghum Beta-glucosidases are chosen as model systems for the study because they each have isozymes exhibiting substrate specificity differences. The approaches of domain-swapping, random and site-directed mutagenesis, in combination with 3-D structure determination and modeling, will be used to accomplish the objective. To define broadly the domains involved in substrate specificity, chimeric Beta-glucosidases are constructed by domain-swapping between two different isozymes. Then cDNAs encoding parental enzymes are subjected to random mutagenesis to generate mutants that are able to hydrolyze a chromogenic or fluorogenic substrate that is not hydrolyzed by parental enzymes. Sequencing DNA of such mutants will identify specific amino acids that play a key role in determining substrate specificity. Finally, specific amino acids that are located at or around the active center and implicated in aglycone recognition are replaced by site-directed mutagenesis individually and stepwise to determine their effects on substrate specificity and kinetic parameters. In addition, the site of aglycone recognition and binding is directly identified using X-ray diffraction of crystallized enzyme-aglycone or enzyme-substrate analogue complexes and modeling.The enzyme Beta-glucosidase plays a key role in many biological processes, which makes it a suitable target for genetic engineering to address problems in agriculture, forestry, and biomass conversion. These processes include chemical defense of plants against pests by producing toxic substances from Beta-glucosides and Beta-glucosinolates; production of simple sugars from large carbohydrates, specifically cellulose (wood), the most abundant substance in the biosphere and a potential source of renewable fuels in the future; and lignin synthesis by trees and the effect of lignin on wood strength and quality for paper production. Thus, the results of the proposed research will have a universal impact on a wide a range of basic and applied research areas dealing with Beta-glucosidases.

本研究的主要目的是了解β -葡萄糖苷酶（β - d -葡萄糖苷葡萄糖水解酶，EC 3.2.1.21）中底物（即苷元）特异性的机制。之所以选择玉米和高粱的β -葡萄糖苷酶作为研究的模型系统，是因为它们各自具有表现出底物特异性差异的同工酶。区域交换、随机和定点突变的方法，结合三维结构确定和建模，将被用来实现这一目标。为了更广泛地定义与底物特异性相关的结构域，嵌合β -葡萄糖苷酶是通过在两种不同的同工酶之间交换结构域来构建的。然后对编码亲本酶的cdna进行随机诱变，产生能够水解不能被亲本酶水解的显色或荧光底物的突变体。这类突变体的DNA测序将确定在确定底物特异性中起关键作用的特定氨基酸。最后，将位于活性中心或其周围与苷元识别有关的特定氨基酸单独或逐步替换为位点定向诱变，以确定其对底物特异性和动力学参数的影响。此外，通过x射线衍射对结晶的酶-苷元或酶-底物类似物进行分析和建模，可以直接确定苷元识别和结合的位点。β -葡萄糖苷酶在许多生物过程中起着关键作用，这使得它成为基因工程解决农业、林业和生物质转化问题的合适目标。这些过程包括通过从β -葡萄糖苷和β -硫代葡萄糖苷中产生有毒物质来对植物进行化学防御；从大碳水化合物，特别是纤维素（木材）生产单糖，这是生物圈中最丰富的物质，也是未来可再生燃料的潜在来源；树木合成木质素及木质素对造纸用木材强度和质量的影响。因此，所提出的研究结果将对β -葡萄糖苷酶的广泛基础和应用研究领域产生普遍影响。