|Ph.D Student||Dalia Shallom|
|Subject||Structure-Function Studies on Alpha-Glucuronidase and|
Alpha-Arabinofuranosidase from Geobacillus
|Department||Department of Biotechnology and Food Engineering||Supervisor||Full Professor Shoham Yuval|
In this study, the glycoside-hydrolases a-D-glucuronidase (AguA) and a-L-arabinofuranosidase (AbfA) from the thermophilic bacterium Geobacillus stearothermophilus were subjected to detailed biochemical characterization and in-depth structural analysis. These enzymes remove methyl-glucuronic acid and arabinofuranosyl side-chains from xylan, and play a crucial role in the natural degradation of this polysaccharide, the most abundant hemicellulose in plants cell-wall.
AguA is a family 67 glycoside-hydrolase which performs the hydrolysis via a single-displacement inverting mechanism. The catalytic residues of AguA were identified by mutagenesis experiments, and the mechanism of catalysis was elucidated from high-resolution (1.5-2.0 Å) crystal structures of the enzyme in complex with substrate and products. AguA was found to have a unique mechanism, in which two residues (Asp364 and Glu392) function as catalytic bases and activate together a nucleophilic water molecule, and the protonated state of the catalytic acid (Glu285) is presumably stabilized by the proximity of the charged substrate.
AbfA is a family 51 glycoside-hydrolase which cleaves the glycosidic bond using a double-displacement retaining mechanism. The catalytic residues of AbfA were unequivocally identified as Glu175 (the acid-base catalyst) and Glu294 (nucleophilic residue) by biochemical characterization of wild-type and mutant enzymes, including detailed kinetic analysis with substrates bearing different leaving groups, pH-dependence activity profiles, and azide rescue experiments. High-resolution (1.2-2.0 Å) crystal structures of AbfA Michaelis complexes with synthetic and natural substrates and of the covalent arabinofuranosyl-enzyme intermediate were analyzed, and the amino-acids residues responsible for substrate binding, catalysis, transition-state stabilization and substrate specificity were determined.