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 66amino 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. The acceptor substrate specificity is N-acetyllactosamine (LacNAc) and lactose, with LacNAc as its preferred acceptor. 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-Nfucopentaose (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.

Targeted Fucosylation of Glycans with Engineered Bacterial Fucosyltransferase Variants

“…We synthesized a variety of LN1 and LN2 and the corresponding H‐antigen glycan epitopes (Table 1) to test them as acceptor substrates for α3FucTs (FucTΔ52‐4M, FucTΔ66). FucTΔ66 from H. pylori NCTC11639 was previously shown to synthesize a series of fucosylated HMOs containing LN2, lacto‐ N ‐ neo tetraose (LN n T), and lacto‐ N ‐ neo hexaose [4b] . LN2‐LN2, LN1‐LN1, and mixed‐type LNs (Table 1) have never been tested yet.…”

“…FucTΔ52‐4M and FucTΔ66 have been used in various studies for the production of HMOs [4b,17,21] or analyses of enzyme structure and mechanism [8] . Both enzymes are described as α3FucTs and showed no α4‐fucosylation activity with LN1 acceptor substrates in previous studies [10,13,15a] .…”

“…While α3FucTs glycosylate only lactose and LN2, [4] α3/4FucTs can also modify LN1 [5] . Both FucTs are able to fucosylate internal LN2 subunits of human milk oligosaccharides and poly‐LN [4b,6] . Strains of the genus Helicobacter pylori are known for their repertoire of different FucTs.…”

“…Subsequent iterative saturation mutagenesis of FucTΔ52 gave the variant FucTΔ52A128N/H129E/Y132I/S46F (FucTΔ52‐4M) with nearly 8‐fold higher activity for the fucosylation of LN2 to produce Le x [12] . These and other H. pylori FucTs were used for the synthesis of fucosylated HMOs with lactose‐based oligo‐ and polysaccharides as substrates [4b,14] . The truncated FucTs FucTΔ66 from H. pylori NCTC11639 [10] and FucTΔ52 from H. pylori 26695 were previously reported to lack α4FucT activity with LN1 as a substrate [15]…”

Effective Separation of Human Milk Glycosides using Carbon Dioxide Supercritical Fluid Chromatography

“…The enzymatic synthesis of fucosylated oligosaccharides by using bacterial fucosyltransferases is considered a robust approach due to the strict regioselectivity and stereoselectivity, broad substrate tolerance, and inherently promiscuous substrate specificity of the enzymes [50] . Thus, bacterial fucosyltransferases are attractive biocatalysts for the preparation of a glycan with multiple fucosylation [45,46,51,52] . However, distinct real‐time identification of individual fucosylated regioisomers enzymatically produced through conventional TLC and HPLC methods remains challenging.…”