|M.Sc Student||Leon Maya|
|Subject||Structure-Function Studies of a Family 43 beta-D-xylosidase|
from Geobacillus stearothemophilus T-6
|Department||Department of Biotechnology||Supervisor||Professor Yuval Shoham|
|Full Thesis text - in Hebrew|
The purpose of this research was to study the structure-function relationship of a family 43 b-D-xylosidase from the thermophilic bacterium Geobacillus stearothermophilus T-6. This enzyme is a part of an array of hemicellulases responsible for the complete degradation of xylan, the most abundant hemicellulosic polymer. b-D-Xylosidases hydrolyze short xylooligosaccharides into xylose units. Enzymes in family 43 use an inverting single-displacement mechanism which is mediated by three conserved carboxylic acids, a general acid, a general base and a pKa modulator. XynB3 was most active at 65°C and pH 6.5, with clear preference to xylose-based substrates. Products analysis indicated that XynB3 is an exoglycosidase that cleaves single xylose units from the non-reducing end of xylooligomers. On the basis of sequence homology, amino acids Asp15, Glu187 and Asp128 were suggested to act as the general-base, general-acid, and the pKa modulator, respectively. These residues were replaced to Ala or Gly residues by site-directed mutagenesis, and the catalytic properties of the resulting mutants (E187G, D15G, D158G) were tested by a combination of a several independent techniques. These techniques included detailed kinetic analysis with substrates bearing different leaving groups, pH-dependence activity profiles, and azide rescue of activity. Kinetic analysis with substrates bearing different leaving groups showed that, for the wild-type enzyme, the kcat and kcat/Km values were only marginally affected by the leaving-group reactivity, whereas for the E187G mutant, both values exhibited significantly greater dependency on the pKa of the leaving group. The pH-dependence activity profile of the putative general acid mutant (E187G) revealed that the protonated catalytic residue was removed. Addition of the exogenous nucleophile azide did not affect the activities of the wild type or the E187G mutant but rescued the activity of the D15G mutant. On the basis of thin-layer chromatography and 1H-NMR analyses, xylose and not xylose azide was the only product of the accelerated reaction, suggesting that the azide ion does not attack the anomeric carbon directly but presumably activates a water molecule. The results provided the first unequivocal evidence regarding the exact role of each of the catalytic residues in an inverting GH43 glycosidases. The structure of XynB3 was solved by molecular replacement revealing a five-bladed b-propeller fold.