Characterization of Helicobacter pylori α1,2‐Fucosyltransferase for Enzymatic Synthesis of Tumor‐Associated Antigens

The α1,2‐fucosyltransferase (α1,2‐FucT) from Helicobacter pylori catalyzes the fucosylation of acceptor oligosaccharides at the C2‐OH of terminal Galβ units. The enzyme from strain NCTC11639 was evaluated for its ability to synthesize cancer‐associated antigens. The α1,2‐FucT was determined to be active over a pH range between 4.0 and 8.0 with the optimum occurring at pH 5.0. Although a divalent metal ion cofactor was not required for catalysis, enhancement of the enzyme activity was detected upon supplement with Mn 2+ . Detailed substrate specificity analysis revealed that α1,2‐FucT can catalyze the fucosylation of a wide variety of oligosaccharide substrates. The α1,2‐FucT preferentially fucosylated type 1 structure (Galβ1‐3GlcNAc)‐containing glycans over type 2 structure (Galβ1‐4GlcNAc)‐containing glycans. The Lewis a trisaccharide [Galβ1‐3(Fucα1‐4)GlcNAc] was found to be the best acceptor. The only exception was that the Lewis x pentasaccharide LNFP III [Galβ1‐4(Fucα1‐3)GlcNAcβ1‐3Galβ1‐4Glc] was favored over the Lewis a analogue LNFP II [Galβ1‐3(Fucα1‐4)GlcNAcβ1‐3Galβ1‐4Glc]. Furthermore, the enzyme exhibited high levels of fucosylation with the type 3/4 disaccharide (Galβ1‐3GalNAc). Enzymatic hydrolysis of the donor substrate, guanosine‐5′‐diphospho‐β‐ L ‐fucose (GDP‐fucose), could be observed in the absence of oligosaccharide substrate. This hydrolysis was completely shifted to an efficient fucosyl transfer in the presence of a substrate sugar. Finally, the H. pylori α1,2‐FucT was successfully used in combination with its α1,3‐FucT counterpart to synthesize the Lewis y tetrasaccharide [Fucα1‐2Galβ1‐4(Fucα1‐3)GlcNAc] from LacNAc (Galβ1‐4GlcNAc) in a milligram scale one‐pot reaction.