טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentZehavi Arie
SubjectBiochemical Characterization and Structure-Function
Analysis of 6-phospho-beta-glycosidases from
Geobacillus Stearothermophilus
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yuval Shoham
Full Thesis textFull thesis text - English Version


Abstract

The degradation of cellulose and hemicellulose, the main components of the plant cell wall, is a pivotal step in the Earth’s carbon cycle. The hydrolysis of plant biomass to soluble sugars requires the synergistic action of several glycoside hydrolases, a widespread group of enzymes that hydrolyze the glycosidic bond between carbohydrates or between a carbohydrate and a non-carbohydrate moiety. Geobacillus stearothermophilus is a thermophilic Gram-positive soil bacterium that possesses an extensive system for utilization of plant cell-wall polysaccharides including xylan, arabinan and galactan. The bacterium can also utilize disaccharides and such two new genes were recently been identified - gan1D encoding for a GH1 6-phospho-β-galactosidase and cel1A encoding for a GH1 6-phospho-β-glucosidase. In this study, we biochemically characterized Gan1D and Cel1A, determined their substrate specificity, identified the catalytic residues of Gan1D and analyzed the three-dimensional structure of Gan1D.

The gan1D gene is a part of the galactan utilization gene cluster encoding for several proteins: a transcriptional regulator (GanR2), a GH4 6-phospho-glucosidase (Gan4C), a three-component regulatory system for sensing galactose (GanPST) and an ABC galactose transport system (GanE2F2G2). The GH1 6-phospho-β-galactosidase, Gan1D, exhibited similar specificity toward several aryl glycosides and β-linked disaccharides with glucose-6-phosphate or galactose-6-phosphate at the glycon moiety. It is likely that the native substrate of Gan1D is galactobiose-6-phosphate, which is the outcome of transporting galactobiose into the cell by a dedicated phosphoenolpyruvate - carbohydrate phosphotransferase system (PTS). Gan1D is a retaining glycosidase and its catalytic residues, Glu170 the acid-base and Glu378 the nucleophile, were identified by detailed kinetic analysis including the azide rescue methodology and pH-dependence activity profiles.

The crystal structure of Gan1D was determined as native and in complex with its substrate and products at high-resolution (1.33-2.21 Å). Based on the crystal structures, the amino-acids residues responsible for substrate binding and specificity were determined. Trp433 was shown to play a functional role in the substrate preference of Gan1D. Replacement of the tryptophan residue to alanine or methionine shifted the substrate preference of the enzyme toward substrates with glucose-6-phosphate at the glycon moiety.

The cel1A gene is a part of the cellobiose utilization gene cluster encoding for a phosphotransferase system (CelBCD) and a transcriptional regulator (CelR).  Cel1A was active toward several synthetic and natural substrates, which contained glucose-6-phosphate but not galactose-6-phosphate at the glycon moiety. Analysis of Cel1A structural model suggested that the enzyme could interact with the phosphate group of glucose-6-phophate, but not with that of galactose-6-phosphate.