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

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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.