Biochemical characterization and molecular cloning of an alpha-1,2-fucosyltransferase that catalyzes the last step of cell wall xyloglucan biosynthesis in pea

Pea microsomes contain an alpha-fucosyltransferase that incorporates fucose from GDP-fucose into xyloglucan, adding it preferentially to the 2-O-posit ion of the galactosyl residue closest to the reducing end of the repeating subunit, This enzyme was solubilized with detergent and purified by affinit y chromatography on GDP-hexanolamine-agarose followed by gel filtration. By utilizing peptide sequences obtained from the purified enzyme, a cDNA clon e was isolated that encodes a 565-amino acid protein with a predicted molec ular mass of 64 kDa and shows 62.3% identity to its Arabidopsis homolog. Th e purified transferase migrates at similar to 63 kDa by SDS-polyacrylamide gel electrophoresis but elutes from the gel filtration column as an active protein of higher molecular weight (similar to 250 kDa), indicating that th e active form is an oligomer, The enzyme is specific for xyloglucan and is inhibited by xyloglucan oligosaccharides and by the by-product GDP, The enz yme has a neutral pH optimum and does not require divalent ions. Kinetic an alysis indicates that GDP-fucose and xyloglucan associate with the enzyme i n a random order. N-Ethylmaleimide, a cysteine-specific modifying reagent, had little effect on activity, although several other amino acid-modifying reagents strongly inhibited activity.