title: Purification and characterization of β-xylosidase that is active for plant complex type N-glycans from tomato (Solanum lycopersicum): removal of core α1-3 mannosyl residue is prerequisite for hydrolysis of β1-2 xylosyl residue.
abstract: In this study, we purified and characterized the β-xylosidase involved in the turnover of plant complex type N-glycans to homogeneity from mature red tomatoes. Purified β-xylosidase (β-Xyl'ase Le-1) gave a single band with molecular masses of 67 kDa on SDS-PAGE under a reducing condition and 60 kDa on gelfiltration, indicating that β-Xyl'ase Le-1 has a monomeric structure in plant cells. The N-terminal amino acid could not be identified owing to a chemical modification. When pyridylaminated (PA-) N-glycans were used as substrates, β-Xyl'ase Le-1 showed optimum activity at about pH 5 at 40 °C, suggesting that the enzyme functions in a rather acidic circumstance such as in the vacuole or cell wall. β-Xyl'ase Le-1 hydrolyzed the β1-2 xylosyl residue from Man₁Xyl₁GlcNAc₂-PA, Man₁Xyl₁Fuc₁GlcNAc₂-PA, and Man₂Xyl₁Fuc₁GlcNAc₂-PA, but not that from Man₃Xyl₁GlcNAc₂-PA or Man₃Xyl₁Fuc₁GlcNAc₂-PA, indicating that the α1-3 arm mannosyl residue exerts significant steric hindrance for the access of β-Xyl'ase Le-1 to the xylosyl residue, whereas the α1-3 fucosyl residue exerts little effect. These results suggest that the release of the β1-2 xylosyl residue by β-Xyl'ase Le-1 occurs at least after the removal the α-1,3-mannosyl residue in the core trimannosyl unit.
authors.affiliation: Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka 1-1-1, Okayama 700-8530, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yokouchi
authors.forename: Natsuko
authors.initials: N
authors.lastname: Ono
authors.forename: Kosuke
authors.initials: K
authors.lastname: Nakamura
authors.forename: Megumi
authors.initials: M
authors.lastname: Maeda
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Kimura
mesh_terms: D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005638:Fruit / Q000737:chemistry* / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D018551:Solanum lycopersicum / Q000737:chemistry* / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000737:chemistry*; D013379:Substrate Specificity; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D011134:Polysaccharides; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase