Functional purification and characterization of a GDP-fucose: beta-N-acetylglucosamine (Fuc to Asn linked GlcNAc) alpha 1,3-fucosyltransferase from mung beans.
An alpha 1,3-fucosyltransferase was purified 3000-fold from mung bean seedlings by chromatography on DE 52 cellulose and Affigel Blue, by chromatofocusing, gelfiltration and affinity chromatography resulting in an apparently homogenous protein of about 65 kDa on SDS-PAGE. The enzyme transferred fucose from GDP-fucose to the Asn-linked N-acetylglucosaminyl residue of an N-glycan, forming an alpha 1,3-linkage. The enzyme acted upon N-glycopeptides and related oligosaccharides with the glycan structure GlcNAc2Man3 GlcNAc2. Fucose in alpha 1,6-linkage to the asparagine-linked GlcNAc had no effect on the activity. No transfer to N-glycans was observed when the terminal GlcNAc residues were either absent or substituted with galactose. N-acetyllactosamine, lacto-N-biose and N-acetylchito-oligosaccharides did not function as acceptors for the alpha 1,3-fucosyltransferase. The transferase exhibited maximal activity at pH 7.0 and a strict requirement for Mn2+ or Zn2+ ions. The enzyme's activity was moderately increased in the presence of Triton X-100. It was not affected by N-ethylmaleimide.
Staudacher
E
E
Institut für Chemie, Universität für Bodenkultur, Vienna, Austria.
Dalik
T
T
Wawra
P
P
Altmann
F
F
März
L
L
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D007887:Fabaceae / Q000201:enzymology*; D005647:Fucosyltransferases / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000138:chemical synthesis*; D010946:Plants, Medicinal; D011134:Polysaccharides / Q000138:chemical synthesis*
D009844:Oligosaccharides; D011134:Polysaccharides; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase