α-L-fucosyltransferases from radish primary roots

Abstract

A novel α-L-fucosyltransferase capable of transferring L-fucose (L-Fuc) from GDP-L-Fuc to the O-2 of α-L-arabinofuranosyl residue (GDP-L-Fuc:α-L-arabinofuranoside 2-α-L-fucosyltransferase) has been found in the microsomal fraction of primary roots from 6-d-old radish (Raphanus sativus L.) seedlings. Enzyme activity was measured fluorometrically at 25°C using a pyridylaminated trisaccharide, L-arabinofuranosylfα(1→3)D-galactopyranosylβ(1→6)D- galactose (AraGalGal-PA) as the acceptor. This enzyme found in the microsomal fraction is maximally active at pH 6.8 and requires 0.1% (w/v) Zwittergent 3-16 and 5 mM Mn2+. Chemical and enzymatic analyses of fucosylated AraGalGal-PA confirmed the attachment of L-Fuc to the L-arabinofuranosyl (L-Araf) residue at O-2 by of-glycosidic linkage. Radiolabeling was used to assay L-Fuc transfer to L-Araf-containing galacto-oligomers and tamarind xyloglucan. The enzyme specific for the L-Araf residue undergoes development-and organ-specific expression in root tissue, whereas the L-Fuc transfer to tamarind xyloglucan can be detected in microsomal fractions from various organs in developing radish plants. Enzyme assays of membranes fractionated from microsomal fractions revealed that two distinct α-L-fucosyltransferases with different acceptor specificity are associated with Golgi membranes from primary roots, whereas hypocotyl Golgi membranes completely lack the enzyme specific for the L-Araf residue.