Glycobiology Advance Access published online on January 12, 2004
Glycobiology, doi:10.1093/glycob/cwh053
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© 2004 Oxford University Press
ORIGINAL ARTICLES
1 Unité de Génétique Moléculaire Animale, UMR 1061 Université-INRA, GDR-CNRS 2590, Institut des Sciences de la Vie et de la Santé, Faculté des Sciences et Techniques, 87060 Limoges, France All vertebrate
Revised on December 30, 2003
Accepted on December 30, 2003
Structure/function study of Lewis
3- and 3/4-fucosyltransferases: the 1,4 fucosylation requires an aromatic residue in the acceptor-binding domain
3- and 3/4-FUTs possess the characteristic acceptor-binding motif "VxxHH(W/R)(D/E)". FUT6 and FUTb enzymes, harboring R in the acceptor-binding motif, transfer fucose in 1,3 linkage, while FUT3 and FUT5 enzymes with W at the candidate position can also transfer fucose in 1,4 linkage - FUT3 being more efficient than FUT5. To determine the involvement of the W/R residue in acceptor recognition, we produced thirty-four variants of human FUT3, FUT5, FUT6 and ox FUTb Lewis enzymes. Among the FUT3 variants where W111 was replaced by the other amino acids, only enzymes with an aromatic residue at the candidate position kept about 50% of 1,4 activity and showed no changes in Km values for GDP-Fuc donor and H-type 1 acceptor substrates. All other substitutions produced enzymes with less than 20% of the 1,4 activity. Thus, the ability of 3/4-FUTs to recognize type 1 substrates involves the aromatic character of W in the acceptor-binding domain. The 1,3 activity of FUT6 and FUTb significantly decreased when their R residue was substituted by basic or charged residues. Moreover, FUT3 and FUT5 variants with W
R substitution had a better affinity for H-type 2 substrate and higher 1,3 activities. Therefore, the optimal fucose addition in 1,3 linkage requires the R residue in the acceptor-binding motif of Lewis FUTs.
Aromatic amino acid, Acceptor substrate specificity, Conserved peptide motifs, Lewis fucosyltransferase, Site-directed mutagenesis
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