5 years ago

Endo-α-Mannosidase-Catalyzed Transglycosylation

Endo-α-Mannosidase-Catalyzed Transglycosylation
Kiichiro Totani, Akira Seko, Yuta Kasahara, Yoichi Takeda, Yukishige Ito, Ichiro Matsuo, Yayoi Yoshimura, Shogo Iwamoto
In order for facilitating the synthesis of oligosaccharides, transglycosylation reactions mediated by glycoside hydrolases have been studied in various contexts. In this study, we examined the transglycosylating activity of a Golgi endo-α-mannosidase. We prepared various glycosyl donors and acceptors, and recombinant human Golgi endo-α-mannosidase and its various mutants were expressed. The enzyme was able to mediate transglycosylation from α-glycosyl-fluorides. Systematic screening of various point mutants revealed that the E407D mutant had excellent transglycosylation activity and extremely low hydrolytic activity. Substrate specificity analysis revealed that minimum motif required for glycosyl acceptor is Manα1– 2Man. The synthetic utility of the enzyme was demonstrated by generation of a high-mannose-type undecasaccharide (Glc1Man9GlcNAc2). Golgi endo-α-mannosidase (G-EM) as a trans-glycosylation catalyst: Transglycosylation mediated by glycoside hydrolases is valuable for the chemo-enzymatic synthesis of oligosaccharides. We developed a novel transglycosylation reaction mediated by a mutant of G-EM (E407D) with α-fluorinated glucosyl mannoside as a donor. This reaction was applied to the synthesis of a natural high-mannose-type dodecasaccharide.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/cbic.201700111

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