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.