Reaction mechanism and substrate specificity for nucleotide sugar of mammalian alpha1,6-fucosyltransferase--a large-scale preparation and characterization of recombinant human FUT8.
FUT8, mammalian alpha1,6-fucosyltransferase, catalyzes the transfer of a fucose residue from the donor substrate, guanosine 5'-diphosphate (GDP)-beta-L-fucose, to the reducing terminal GlcNAc of the core structure of asparagine-linked oligosaccharide via an alpha1,6-linkage. FUT8 is a typical type II membrane protein, which is localized in the Golgi apparatus. We have previously shown that two neighboring arginine residues that are conserved among alpha1,2-, alpha1,6-, and protein O-fucosyltransferases play an important role in donor substrate binding. However, details of the catalytic and reaction mechanisms and the ternary structure of FUT8 are not understood except for the substrate specificity of the acceptor. To develop a better understanding of FUT8, we established a large-scale production system for recombinant human FUT8, in which the enzyme is produced in soluble form by baculovirus-infected insect cells. Kinetic analyses and inhibition studies using derivatives of GDP-beta-L-fucose revealed that FUT8 catalyzes the reaction which depends on a rapid equilibrium random mechanism and strongly recognizes the base portion and diphosphoryl group of GDP-beta-L-fucose. These results may also be applicable to other fucosyltransferases and glycosyltransferases.
Ihara
Hideyuki
H
Department of Biochemistry, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
Ikeda
Yoshitaka
Y
Taniguchi
Naoyuki
N
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002384:Catalysis; D005643:Fucose / Q000737:chemistry; D005647:Fucosyltransferases / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D025942:Oligosaccharides, Branched-Chain / Q000138:chemical synthesis / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity
D025942:Oligosaccharides, Branched-Chain; D011994:Recombinant Proteins; D005643:Fucose; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase