The engineered hexosaminidase TtOGA-D120N is an efficient O-/N-/S-glycoligase that also catalyzes formation and release of oxazoline donors for cascade syntheses with glycosynthases or transglycosylases

Engineering glycoside hydrolases is a major route to obtain catalysts forming glycosidic bonds. Glycosynthases, thioglycoligases and transglycosylases represent the main strategies, each having advantages and drawbacks. Here, we show that an engineered enzyme from the GH84 family, the acid-base mutant TtOGA-D120N, is an efficient O-, N- and S-glycoligase, able to use S_(sp^3 ), O_(sp^3 ), N_(sp^2 ), and N_sp nucleophiles. Moreover, TtOGA-D120N catalyzes the formation and release of N-acetyl-d-glucosamine 1,2-oxazoline, the intermediate of hexosaminidases displaying substrate-assisted catalysis. This release of an activated intermediate allows cascade synthesis by combination with transglycosylases or glycosynthases, here exemplified by synthesis of the human milk oligosaccharide lacto-N-triose II.