A single amino acid in the hypervariable stem domain of vertebrate alpha1,3/1,4-fucosyltransferases determines the type 1/type 2 transfer. Characterization of acceptor substrate specificity of the lewis enzyme by site-directed mutagenesis.
Alignment of 15 vertebrate alpha1,3-fucosyltransferases revealed one arginine conserved in all the enzymes employing exclusively type 2 acceptor substrates. At the equivalent position, a tryptophan was found in FUT3-encoded Lewis alpha1,3/1,4-fucosyltransferase (Fuc-TIII) and FUT5-encoded alpha1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in alpha1, 4-linkage. The single amino acid substitution Trp111 --> Arg in Fuc-TIII was sufficient to change the specificity of fucose transfer from H-type 1 to H-type 2 acceptors. The additional mutation of Asp112 --> Glu increased the type 2 activity of the double mutant Fuc-TIII enzyme, but the single substitution of the acidic residue Asp112 in Fuc-TIII by Glu decreased the activity of the enzyme and did not interfere with H-type 1/H-type 2 specificity. In contrast, substitution of Arg115 in bovine futb-encoded alpha1, 3-fucosyltransferase (Fuc-Tb) by Trp generated a protein unable to transfer fucose either on H-type 1 or H-type 2 acceptors. However, the double mutation Arg115 --> Trp/Glu116 --> Asp of Fuc-Tb slightly increased H-type 1 activity. The acidic residue adjacent to the candidate amino acid Trp/Arg seems to modulate the relative type 1/type 2 acceptor specificity, and its presence is necessary for enzyme activity since its substitution by the corresponding amide inactivated both Fuc-TIII and Fuc-Tb enzymes.
Dupuy
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Institut de Biotechnologie, Faculté des Sciences, Université de Limoges, 123 Avenue Albert Thomas, 87060 Limoges, Université de Paris Sud XI, 94807 Villejuif Cedex, France.
Petit
J M
JM
Mollicone
R
R
Oriol
R
R
Julien
R
R
Maftah
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002417:Cattle; D017931:DNA Primers; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase