Glycobiology Advance Access originally published online on December 11, 2005
Glycobiology 2006 16(4):333-342; doi:10.1093/glycob/cwj068
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Reaction mechanism and substrate specificity for nucleotide sugar of mammalian 
1,6-fucosyltransferase—a large-scale preparation and characterization of recombinant human FUT8
2 Department of Biochemistry, Osaka University Graduate School of Medicine, B1, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; 3 Division of Molecular Biology, Department of Biomolecular Sciences, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan; and 4 Core Research for Evolution Science and Technology, Japanese Science and Technology Agency, 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan JST
1 To whom correspondence should be addressed; e-mail: proftani{at}biochem.med.osaka-u.ac.jp
Received on October 12, 2005; revised on December 1, 2005; accepted on December 7, 2005
FUT8, mammalian 
1,6-fucosyltransferase, catalyzes the transfer of a fucose residue from the donor substrate, guanosine 5'-diphosphate (GDP)-ß-L-fucose, to the reducing terminal GlcNAc of the core structure of asparagine-linked oligosaccharide via an 1,6-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 1,2-, 1,6-, 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-ß-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-ß-L-fucose. These results may also be applicable to other fucosyltransferases and glycosyltransferases.
Key words: fucosyltransferase / FUT8 / baculovirus-insect cell expression system / kinetic analysis / reaction mechanism
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