Fucosylation is an important glycan modification that is critical for immune recognition, microbial interactions, and cancer metastasis. For example, terminal fucosylation (α1,2/3/4) is characteristic of the histo-blood group antigens (ABO, H, and Lewis), and fucosylated human milk oligosaccharides (HMOs) impart advantageous prebiotic, antimicrobial, and immunomodulating effects. Given these biological implications, highly efficient synthetic methods to access diverse fucosylated oligosaccharides are invaluable for detailed structure-function relationship studies and therapeutic applications. Here, we report the design and application of α-glycoligases generated from Bifidobacterium bifidum’s retaining α1,3/4-fucosidase (AfcB) that enable efficient α1,3/4-fucosylation using a simple α-fucosyl-fluoride donor substrate. Substitution of AfcB’s general acid/base (E746) with several other amino acids including Ala, Gly, Ser, and Gln, 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 the Lewis X and Lewis A trisaccharides, 3-fucosyllactose, and lacto-N-fucopentaose III (two fucosylated HMOs) in high yield with only slight excess (1.5 eq.) of α-fucosyl-fluoride. The α-fucoligase demonstrates a unique preference for a reducing end N-acetylglucosamine over glucose in its acceptor substrate, preferentially synthesizes α1,3- over α1,4-fucosidic bonds, and appears to require a free terminal galactose for catalysis on non-reducing residue that is immediately adjacent . This new enzymatic fucosylation method utilizes a simple, readily synthesized, and stable donor substrate, avoiding the need for expensive GDP-fucose and the unstable α-fucosyl-fluoride as the donor substrates for α1,3/4-fucosyltransferase- 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-fucosylated HMOs and other related oligosaccharides.

Fucosylation is an important glycan modification that is critical for immune recognition, microbial interactions, and cancer metastasis. For example, terminal fucosylation (α1,2/3/4) is characteristic of the histo-blood group antigens (ABO, H, and Lewis), and fucosylated human milk oligosaccharides (HMOs) impart advantageous prebiotic, antimicrobial, and immunomodulating effects. Given these biological implications, highly efficient synthetic methods to access diverse fucosylated oligosaccharides are invaluable for detailed structure-function relationship studies and therapeutic applications. Here, we report the design and application of α-glycoligases generated from _Bifidobacterium bifidum_’s retaining α1,3/4-fucosidase_ _(AfcB) that enable efficient α1,3/4-fucosylation using a simple α-fucosyl-fluoride donor substrate. Substitution of AfcB’s general acid/base (E746) with several other amino acids including Ala, Gly, Ser, and Gln, 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 the Lewis X and Lewis A trisaccharides, 3-fucosyllactose, and lacto-_N_-fucopentaose III (two fucosylated HMOs) in high yield with only slight excess (1.5 eq.) of α-fucosyl-fluoride. The α-fucoligase demonstrates a unique preference for a reducing end _N_-acetylglucosamine over glucose in its acceptor substrate, preferentially synthesizes α1,3- over α1,4-fucosidic bonds, and appears to require a free terminal galactose for catalysis on non-reducing residue that is immediately adjacent . This new enzymatic fucosylation method utilizes a simple, readily synthesized, and stable donor substrate, avoiding the need for expensive GDP-fucose and the unstable α-fucosyl-fluoride as the donor substrates for α1,3/4-fucosyltransferase- 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-fucosylated HMOs and other related oligosaccharides.

Engineering a novel α-glycoligase for chemoenzymatic synthesis of α1,3/4-fucosylated oligosaccharides

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