1,2-alpha-l-Fucosynthase: a glycosynthase derived from an inverting alpha-glycosidase with an unusual reaction mechanism.

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucalpha1,2Gal linkage by introducing glycosynthase technology into 1,2-alpha-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 beta-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2'-fucosyllactose. 1,2-alpha-l-Fucosynthase is the first glycosynthase derived from an inverting alpha-glycosidase and from a glycosidase with an unusual reaction mechanism.