|Ph.D Student||Bravman Tsafrir|
|Subject||The Catalytic Mechanism of beta-Xylosidases from|
Families 39 and 52 Glycoside Hydrolases
|Department||Department of Biotechnology and Food Engineering||Supervisors||Professor Yuval Shoham|
|Professor Emeritus Uri Cogan|
b-D-Xylosidases (EC 18.104.22.168) are glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. Together with other hemicellulolytic enzymes, they take part in the complete degradation of xylan, a major polysaccharide in the plant cell wall. Glycoside hydrolases are capable of accelerating the hydrolysis of the glycosidic bond up to 1017-fold, making them of the most proficient catalysts in nature. The hydrolysis takes place via general acid catalysis that requires usually two carboxylic acids: a proton donor and a nucleophile/base, and may result in either retention or inversion of the configuration of the anomeric carbon.
In this study two b-xylosidases genes, xynB1 and xynB2, from the thermophilic bacterium Geobacillus stearothermophilus T-6, were cloned, overexpressed in Escherichia coli, and their gene products were purified to homogeneity. Based on sequence alignment, XynB1 and XynB2 belong to family 39, and 52 of glycoside hydrolases (GH), respectively. The stereochemical course of hydrolysis of XynB2 was followed by 1H-NMR and the spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 GH. The two catalytic residues of XynB2 were identified through detailed kinetic analysis using substrates bearing different leaving groups, chemical rescue, and pH dependence profiles. Glu335 and Asp495 were identified as the catalytic nucleophile and acid-base, respectively, of the family 52 XynB2. Similarly, Glu160 was identified as the catalytic acid-base of XynB1 from family 39 GH.
The nucleophile mutant of XynB2 was shown to function as an effective glycosynthase, synthesizing various xylooligosaccharaides. The products formed were stable towards hydrolysis by the mutant enzyme.