Exploring the Synthetic Application of Helicobacter pylori α1,3/4-Fucosyltransferase FucTIII toward the Syntheses of Fucosylated Human Milk Glycans and Lewis Antigens

Abstract

In this study, we focused on the synthetic application of α1,3/4-fucosyltransferase obtained from Helicobacter pylori DSM 6709 (FucTIII) on l-fucose-containing glycans. By combining FucTIII with the sequential one-pot enzymatic system of human milk oligosaccharide (HMO) production, various fucosylated HMOs, such as lacto-N-fucopentose V (LNFP V), LNFP VI, lacto-N-difucohexaose II (LNDFH II), and lacto-N-neodifucohexaose II (LNnDFH II), were synthesized. Moreover, l-fucose-containing glycan synthesis of Lewis antigens, such as Lewis x, Lewis y, Lewis a, Lewis b, sialyl Lewis x, sialyl Lewis a, and their derivatives, was achieved. Enzyme kinetics proved that the catalytic efficiency (k cat/K m) of FucTIII on type-2 N-acetyl lactosamine (LacNAc) was 39 times higher than that on type-1 LacNAc. Furthermore, enzyme kinetics revealed that additional GlcNAc on the nonreducing end of the acceptors can enhance the catalytic efficiency of FucTIII on the glycan acceptors. Investigations of bacterial FucTs on substrate spectra indicate that future studies should study expansion of the inventory of biocatalysis for the synthesis of valuable glycans.

Targeted Fucosylation of Glycans with Engineered Bacterial Fucosyltransferase Variants

“…GlcNAc‐terminated saccharides are interesting substrates for targeted fucosylation of the LN subunit at the reducing end because a single terminal GlcNAc does not function as an acceptor for fucosylation. The α3/4FucT from H. pylori DSM 6709 can fucosylate LNT II as well as the LN‐based trisaccharides (GlcNAc‐LN2, GlcNAc‐LN1) [5a] . In the present study, both FucTs fucosylated the reducing GlcNAc of the trisaccharides (Figure 2C, Figure S15).…”

“…HPLC analysis of the fucosidase‐treated glycan with the untreated glycan showed no shift in the retention time of the compound (Figure S16). In contrast to the α3/4FucT from H. pylori DSM 6709, [5a] FucTΔ66 and FucTΔ52‐4M showed a higher conversion for the glycan GlcNAcβ,3LN1. However, both enzymes showed unexpected α4FucT activity.…”

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

“…This study employed SFC coupled with both evaporative light scattering detectors and UV‐vis detectors (see Supporting Information Figure S1). To systematically access the potential of SFC for complex oligosaccharide analysis and separation, a series of well‐defined oligosaccharides (Figure 1), all featuring a lactose core structure with an azidohexyl linker at the reducing‐end, were prepared using enzymatic glycan synthesis according to the method described in our other work [44–46] …”

“…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.…”

“…However, distinct real‐time identification of individual fucosylated regioisomers enzymatically produced through conventional TLC and HPLC methods remains challenging. To explore the applicability of SFC to fucosylated regioisomer discrimination, multiple fucosylation on a type‐1 pentasaccharide ( 4 ) catalyzed by FucTIII (α1,3/4‐fucosyltransferase from Helicobacter pylori DSM 6709) [45] was performed and analysis conducted. FucTIII could be used for synthesizing two monofucosylated products, GlcNAcβ1,3‐LNFP II ( 13 ) and GlcNAcβ1,3‐LNFP V ( 14 ), and the difucosylated product GlcNAcβ1,3LNDFH II ( 15 ) in one reaction.…”