A single amino acid in the hypervariable stem domain of vertebrate α1,3/1,4-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 alpha 1,3-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 alpha 1,3/1,4-fucosyltransferase (Fuc-TIII) and FUT5-encoded alpha 1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in alpha 1,4-linkage, The single amino acid substitution Trp(111) --> Arg in Fuc-TIII was sufficient to change the specificity of fucose transfer from H-type 1 to H-type 2 accepters. The additional mutation of Asp(112) --> Glu increased the type 2 activity of the double mutant Fuc-TIII enzyme, but the single substitution of the acidic residue Asp(112) 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 Arg(115) in bovine futb-encoded alpha 1,3-fucosyltransferase (Fuc-Tb) by Trp generated a protein unable to transfer fucose either on H-type 1 or H-type 2 accepters. However, the double mutation Arg(115) --> TrP/Glu(116) --> 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.