1,2-alpha-l-Fucosynthase: a glycosynthase derived from an inverting alpha-glycosidase with an unusual reaction mechanism |
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Authors: | Wada Jun Honda Yuji Nagae Masamichi Kato Ryuichi Wakatsuki Soichi Katayama Takane Taniguchi Hajime Kumagai Hidehiko Kitaoka Motomitsu Yamamoto Kenji |
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Affiliation: | Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan. |
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Abstract: | 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. |
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Keywords: | ESI-MS, electrospray ionization-mass spectrometry Fuc, smallcaps" >l-fucose FucF, β- smallcaps" >l-fucosyl fluoride 2′-FL, 2′-fucosyllactose Gal, galactose GH, glycoside hydrolase GlcNAc, N-acetylglucosamine Lac, lactose MOPS, 3-morpholinopropanesulfonic acid NMR, nuclear magnetic resonance |
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