Glycoside Hydrolase Family 39 β-Xylosidases Exhibit β-1,2-Xylosidase Activity for Transformation of Notoginsenosides: A New EC Subsubclass.

β-1,2-Xylosidase activity has not been recorded as an EC subsubclass. In this study, phylogenetic analysis and multiple sequence alignments revealed that characterized β-xylosidases of glycoside hydrolase family (GH) 39 were classified into the same subgroup with conserved amino acid residue positions participating in substrate recognition. Protein-ligand docking revealed that seven of these positions were probably essential to bind xylose-glucose, which is linked by a β-1,2-glycosidic bond. Amino acid residues in five of the seven positions are invariant, while those in two of the seven positions are variable with low frequency. Both the wild-type β-xylosidase rJB13GH39 and its mutants with mutation at the two positions exhibited β-1,2-xylosidase activity, as they hydrolyzed o-nitrophenyl-β-d-xylopyranoside and transformed notoginsenosides R ₁ and R ₂ to ginsenosides Rg ₁ and Rh ₁ , respectively. The results suggest that all of these characterized GH 39 β-xylosidases probably show β-1,2-xylosidase activity, which should be assigned an EC number with these β-xylosidases as representatives.