|Ph.D Student||Tabachnikov Orly|
|Subject||Protein Engineering and Structure-Function Relationships of|
Enzymes Involved in Galactan Utilization from
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Yuval Shoham|
|Full Thesis text|
The natural degradation of plant cell wall polysaccharides by microorganisms is a key step in the carbon cycle on Earth. The heterogenic structure of these polymers requires synergistic activity of many hydrolytic enzymes, named glycoside hydrolases (GHs), with different substrate specificities.
In this study, a 9.4-kb gene cluster ganREFGBA for galactan-utilization in the thermophilic bacterium Geobacillus stearothermophilus T-6 was characterized. Based on sequence analysis, biochemical characterization and binding measurements, we propose the following pathway for the utilization of galactan in this bacterium: the extracellular galactanase GanA cleaves galactan into galacto-oligosaccharides (mainly galactotetraose), that enter the cell via a specific ATP-binding-cassette transport system GanEFG, where they are further degraded by the intracellular β-galactosidase GanB into galactose monomers and metabolized in the cell. The GanR protein presumably regulates the transcription of the galactan utilization genes. This strategy of producing relatively large oligosaccharides by secreted enzymes has the advantage that the extracellular degradation products are not easily available to non-hemicellulolytic competing microorganisms.
The crystal structure of GanB has been determined at resolution of 2.5 Å. The quaternary structure of GanB is homotrimer, where each subunit is composed of three structural domains. The interactions between subunits form a pocket shaped active site, which is typical to exo-acting enzymes. Moreover, a tryptophan residue, that is located on a loop at the surface of each subunit, can form a hydrogen bond via a water molecule with galactose that is bound at the catalytic site of the next subunit, and thus participates directly in catalysis. A Zn ion that interacts with four conserved cysteine residues contributes to the thermal stability of the protein. Furthermore, this feature contributes to the exact position of the tryptophan in the active site, and therefore affects catalysis.
Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study, we demonstrated that GanB-E323A can function as glycosynthase using α-D-galactopyranosyl- and β-L-arabinopyranosyl fluoride as donors and various aryl sugars as acceptors, creating mainly products with β-1,4 linkages. The mutant enzyme can also catalyze the self-condensation reaction of α-D-galactopyranosyl fluoride, producing mainly α-D-galactobiosyl fluoride. The obtained di- and trisaccharide products may be used for various applications in medicine and food industry.