Identification of the catalytic residues in family 52 glycoside hydrolase, a beta-xylosidase from Geobacillus stearothermophilus T-6.
beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a beta-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased approximately 106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pKa). The mutant exhibited 103-fold reduction in activity, and the Brønsted plot of log(kcat) versus pKa revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (kcat/Km), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pKa) but decreased 102-fold for those that require strong acid catalysis (high pKa). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2.
Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Tsafrir
T
Bravman
Valery
V
Belakhov
Dmitry
D
Solomon
Gil
G
Shoham
Bernard
B
Henrissat
Timor
T
Baasov
Yuval
Y
Shoham
D019943:Amino Acid Substitution; D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019810:Sodium Azide; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D019810:Sodium Azide; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase