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-position of the galactosyl residue closest to the reducing end of the repeating subunit. This enzyme was solubilized with detergent and purified by affinity chromatography on GDP-hexanolamine-agarose followed by gel filtration. By utilizing peptide sequences obtained from the purified enzyme, a cDNA clone was isolated that encodes a 565-amino acid protein with a predicted molecular mass of 64 kDa and shows 62.3% identity to its Arabidopsis homolog. The purified transferase migrates at approximately 63 kDa by SDS-polyacrylamide gel electrophoresis but elutes from the gel filtration column as an active protein of higher molecular weight ( approximately 250 kDa), indicating that the active form is an oligomer. The enzyme is specific for xyloglucan and is inhibited by xyloglucan oligosaccharides and by the by-product GDP. The enzyme has a neutral pH optimum and does not require divalent ions. Kinetic analysis indicates that GDP-fucose and xyloglucan associate with the enzyme in 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.
Faik
A
A
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824, USA.
Bar-Peled
M
M
DeRocher
A E
AE
Zeng
W
W
Perrin
R M
RM
Wilkerson
C
C
Raikhel
N V
NV
Keegstra
K
K
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D005647:Fucosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005936:Glucans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D011134:Polysaccharides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014990:Xylans; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D005647:Fucosyltransferases; D005936:Glucans; D011134:Polysaccharides; D011994:Recombinant Proteins; D014990:Xylans; D000097631:Galactoside 2-alpha-L-fucosyltransferase; C029353:xyloglucan