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

9905698 Esen本研究的主要目的是了解底物的机制（即，糖苷配基）在β-葡糖苷酶（β-D-葡糖苷葡糖水解酶，EC 3.2.1.21）中的特异性。 选择玉米和高粱β-葡萄糖苷酶作为研究的模型系统，因为它们各自具有表现出底物特异性差异的同工酶。 结构域交换，随机和定点突变的方法，结合3-D结构测定和建模，将被用来实现这一目标。为了广泛地定义底物特异性所涉及的结构域，通过两种不同同工酶之间的结构域交换来构建嵌合β-葡糖苷酶。 然后对编码亲本酶的cDNA进行随机诱变以产生能够水解不被亲本酶水解的显色或荧光底物的突变体。 对这些突变体的DNA测序将鉴定在确定底物特异性中起关键作用的特定氨基酸。 最后，位于活性中心或其周围并参与糖苷配基识别的特定氨基酸被单独和逐步的定点诱变取代，以确定其对底物特异性和动力学参数的影响。 此外，通过对酶-糖苷配基或酶-底物类似物复合物的X射线衍射分析和建模，可以直接确定糖苷配基的识别和结合位点。β-葡萄糖苷酶在许多生物过程中起着关键作用，这使其成为基因工程的合适靶点，以解决农业、林业和生物质转化中的问题。 这些过程包括植物通过从β-葡糖苷和β-芥子油苷产生有毒物质来对抗害虫的化学防御;从大碳水化合物，特别是纤维素（木材），生物圈中最丰富的物质和未来可再生燃料的潜在来源生产单糖;树木合成木质素以及木质素对木材强度和纸张生产质量的影响。 因此，所提出的研究结果将对涉及β-葡萄糖苷酶的广泛的基础和应用研究领域产生普遍影响。

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.