The attachment of a single glycan moiety, like α-L-fucose, can have tremendous biological implications. Fucosylation is critical for molecular recognition events in the immune system, microbial interactions, and cancer metastasis. Terminal fucosylation (α1,2/3/4) is characteristic of the histo-blood group antigens (ABO, H, and Lewis). In breast milk, α1,2/3/4-fucosylated human milk oligosaccharides (HMOs) impart advantageous prebiotic, antimicrobial, and immunomodulating activities. Terminal fucosylation is also expressed on endothelial and epithelial cells which is critical for immune cell trafficking and signal transduction. Given the biological implications of terminal fucosylation, a highly efficient synthetic method to access diverse fucosylated oligosaccharides is invaluable for detailed structure-function relationship studies and for therapeutic application. Here, we report the design and application of a novel α-glycoligase generated from Bifidobacterium bifidum’s retaining α1,3/4-fucosidase (AfcB) that enables efficient α1,3/4-fucosylation using a simple α-fucosyl fluoride as the donor substrate. Substitution of AfcB’s general acid/base (E746) with seven amino acids, yielded mutants with diminished hydrolysis but enhanced α1,3/4-transfucosylation activity. Comparative analysis indicated that AfcB E746A was the most efficient mutant, which could efficiently catalyze the synthesis of Lewis X, Lewis A, and 3-fucosyllactose in high yields with only a slight excess (1.5 eq.) of α-fucosyl fluoride as the donor substrate. AfcB E746A could also utilizes lacto-N-neotetraose as an acceptor to synthesize two mono-fucosylated lacto-N-fucopentaose regioisomers. We conclude that AfcB E746A catalyzes stereo- and regiospecific fucosylation. The α-fucoligase demonstrates a unique substrate preference for a reducing end N-acetylglucosamine over glucose in its acceptor substrate and preferentially synthesizes α1,3- over α1,4-fucosidic bonds. This novel α1,3/4-fucoligase utilizes a simple, readily synthesized, and stable donor substrate. The new enzymatic fucosylation method avoids the use of expensive GDP-fucose and the highly unstable β-fucosyl fluoride as the donor substrates for the α1,3/4-fucosyltransferase catalyzed and previously reported fucosynthase-catalyzed fucosylation, respectively. Thus, the new α1,3/4-fucoligase provides an efficient tool for the synthesis of α1,3/4-fucosylatyed HMOs and other related oligosaccharides.

Lai-Xi Wang

Appearances