Biochemical characterization of Helicobacter pylori α1–3-fucosyltransferase and its application in the synthesis of fucosylated human milk oligosaccharides

Abstract Fucosylated human milk oligosaccharides (HMOs) have important biological functions. Enzymatic synthesis of such compounds requires robust fucosyltransferases. A C-terminal 66-amino acid truncated version of Helicobacter pylori α1–3-fucosyltransferase (Hp3FT) is a good candidate. Hp3FT was biochemically characterized to identify optimal conditions for enzymatic synthesis of fucosides. While N -acetyllactosamine (LacNAc) and lactose were both suitable acceptors, the former is preferred. At a low guanosine 5′-diphospho-β-L-fucose (GDP-Fuc) to acceptor ratio, Hp3FT selectively fucosylated LacNAc. Based on these enzymatic characteristics, diverse fucosylated HMOs, including 3-fucosyllactose (3-FL), lacto- N -fucopentaose (LNFP) III, lacto- N -neofucopentaose (LNnFP) V, lacto- N -neodifucohexaose (LNnDFH) II, difuco- and trifuco- para -lacto- N -neohexaose (DF-paraLNnH and TF- para -LNnH), were synthesized enzymatically by varying the ratio of the donor and acceptor as well as controlling the order of multiple glycosyltransferase-catalyzed reactions.