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

本研究的主要目的是了解β-葡萄糖苷酶(β-D-葡萄糖苷酶，EC 3.2.1.21)底物(即苷元)专一性的机制。玉米和高粱β-葡萄糖苷酶被选为研究的模型系统，因为它们都有表现出底物专一性差异的同工酶。将采用结构域交换、随机和定点突变的方法，结合三维结构的确定和建模来实现这一目标。为了更广泛地定义与底物专一性有关的结构域，嵌合的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进行随机突变，产生能够降解不被亲本酶水解的显色底物或荧光底物的突变体。对这些突变体的DNA进行测序将识别在决定底物专一性方面起关键作用的特定氨基酸。最后，将位于活性中心或活性中心周围的与苷元识别有关的特定氨基酸逐个和逐步地用定点突变取代，以确定它们对底物专一性和动力学参数的影响。此外，通过对结晶的酶-苷元或酶-底物类似物的X射线衍射和模拟，可以直接确定苷元的识别和结合位置。β-葡萄糖苷酶在许多生物学过程中发挥着关键作用，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靶点。这些过程包括：通过从β-葡萄糖苷和β-硫代葡萄糖酸盐中产生有毒物质，对植物进行化学防御；从大碳水化合物，特别是纤维素(木材)中生产单糖，纤维素(木材)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，以及木质素对木材强度和质量的影响，用于造纸。因此，拟议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.