Purification and characterization of a xyloglucan oligosaccharide-specific xylosidase from pea seedlings.
An alpha-xylosidase that acts on oligosaccharide fragments of xyloglucan, a plant cell wall polysaccharide, was purified from pea (Pisum sativum) epicotyls that had been treated with an auxin analog. The enzyme had an apparent molecular mass of 85,000 Da according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 79,000 Da according to gel-permeation chromatography under nondenaturing conditions. The purified xylosidase consisted of a series of closely related, enzymatically active proteins with isoelectric points ranging from about pH 7.35 to 7.7; the xylosidases were separated by chromatofocusing. The pH optimum of the mixed xylosidase was 4.9-5.1. The substrate specificity of the xylosidase mixture was determined by purification and structural characterization of the products of treating xyloglucan-oligosaccharide substrates with the enzyme. Characterization of the substrates and products included elution volume from a gel-permeation column, glycosyl residue and glycosyl linkage composition analyses, fast atom bombardment-mass spectrometry, and 1H NMR spectroscopy. The enzyme specifically cleaved only one of the alpha-xylosidic linkages of xyloglucan-oligosaccharide substrates, the one attached to a 6-linked glucosyl residue, not those attached to the 4,6-linked glucosyl residues. The enzyme was unable to cleave the xylosidic linkage of p-nitrophenyl-alpha-D-xylopyranoside or the alpha-xylosidic linkage to C-6 of glucose in the disaccharide isoprimeverose. The enzyme was also unable to release measurable amounts of xylose from large xyloglucan polymers.
O'Neill
R A
RA
Complex Carbohydrate Research Center, University of Georgia, Athens 30602.
Albersheim
P
P
Darvill
A G
AG
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D007887:Fabaceae / Q000201:enzymology; D005936:Glucans; D006026:Glycoside Hydrolases / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D010946:Plants, Medicinal; D011134:Polysaccharides / Q000378:metabolism*; D013379:Substrate Specificity; D014990:Xylans; D014995:Xylosidases / Q000302:isolation & purification / Q000378:metabolism*
D005936:Glucans; D009844:Oligosaccharides; D011134:Polysaccharides; D014990:Xylans; C029353:xyloglucan; D006026:Glycoside Hydrolases; D014995:Xylosidases