期刊
BIOCHEMISTRY
卷 62, 期 16, 页码 2358-2362出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.3c00236
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Engineering glycoside hydrolases is an important approach for obtaining catalysts that can form glycosidic bonds. In this study, an engineered enzyme called TtOGA-D120N from the GH84 family was shown to be an efficient O-, N-, and S-glycoligase, capable of using various nucleophiles. The enzyme also catalyzes the formation and release of an activated intermediate, which allows for cascade synthesis in combination with transglycosylases or glycosynthases, as demonstrated by the synthesis of a specific human milk oligosaccharide lacto-N-triose II.
Engineering glycoside hydrolases is a major route to obtaining 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 Ssp3, Osp3, Nsp2, and Nsp 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.
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