Mycosystema. 2013, 32(5): 846-854.
Glycoside hydrolases of 7-xylosyltaxanes LXYL-P1-1 and LXYL-P1-2 are two bifunctional β-D-xylosidases/ β-D-glucosidases cloned from Lentinula edodes. The two enzymes (identity 97%) could specifically remove the xylosyl residue from 7-xylosyltaxanes such as 7-xylosyl-10-deacetyltaxol (XDT). Bioinformatics method was applied to predict the active center of the two enzymes and preliminarily determined Asp300 as the catalytic nucleophile, Glu529 as the general acid/base catalyst, Asn172-Gly173-Arg174and Lys207-His208 as the two conserved motifs for the substrate binding, respectively. The LXYL-P1-2 mutants, including N172A, G173A, R174A, K207A, H208A, D300N and E529Q, were obtained by means of the site-directed mutagenesis technique, with the yeast Pichia pastoris as the expression host. All the mutants were subjected to the enzyme activity analysis. Results showed that both the β-D-xylosidase and β-D-glucosidase activities of N172A, G173A, R174A, K207A, D300N and E529Q were dramatically decreased or even lost. The β-D-xylosidase activity of H208A was also obviously decreased, but it still kept 98% of the β-D-glucosidase activity. The results preliminarily supported the prediction of the active center and provided an experimental basis for further study of the relationship between the structure and function of the glycoside hydrolases of 7-xylosyltaxanes.
