1,2‐α‐l‐Fucosynthase: A glycosynthase derived from an inverting α‐glycosidase with an unusual reaction mechanism

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucα1,2Gal linkage by introducing glycosynthase technology into 1,2-α-l-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using β-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2′-fucosyllactose. 1,2-α-l-Fucosynthase is the first glycosynthase derived from an inverting α-glycosidase and from a glycosidase with an unusual reaction mechanism.