Transglycosylation of engineered cyclodextrin glucanotransferases as O-glycoligases

Li, Chao; Ahn, Hee-Jeong; Kim, Jin Hyo; Kim, Young-Wan

doi:10.1016/j.carbpol.2013.08.056

Cited by 14 publications

(11 citation statements)

References 36 publications

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“…There are two general glycosidase engineering strategies that may convert a glycosidase into a synthetically useful mutant. One is the glycosynthase concept through site-directed mutagenesis at the critical nucleophilic residue of a retaining glycosidase to generate a mutant that is devoid of hydrolysis activity but can take an activated glycosyl donor (usually a glycosyl fluoride) with an opposite anomeric configuration for transglycosylation. − Glycosynthases derived from several GH family glycosidases have been successfully created using this strategy. − The other is the glycoligase approach, first developed by Withers and co-workers, in which the general acid/base residue of a retaining glycosidase is mutated to eliminate the hydrolysis activity and the enzymatic transglycosylation is enabled by using an activated glycosyl donor with the same anomeric configuration. − For β-glycosynthases derived from the corresponding retaining β-glycosidases, the readily synthesized and relatively stable α-glycosyl fluorides have become the common glycosyl donor substrates. , However, the evaluation of the transglycosylation activity of potential α-fucosynthases usually requires a β-fucosyl fluoride, which is quite unstable in aqueous solution and will be hydrolyzed spontaneously with a half-life of ca. 20 min .…”

Section: Resultsmentioning

confidence: 99%

Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

Zhu

Christopher

et al. 2017

J. Am. Chem. Soc.

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Core fucosylation of N-glycoproteins plays a crucial role in modulating the biological functions of glycoproteins. Yet, the synthesis of structurally well-defined, core-fucosylated glycoproteins remains a challenging task due to the complexity in multi-step chemical synthesis or the inability of the biosynthetic α1,6-fucosyltransferase (FUT8) to directly fucosylate full-size mature N-glycans in a chemoenzymatic approach. We report in this paper the design and generation of potential α1,6-fucosynthase and fucoligase for direct core-fucosylation of intact N-glycoproteins. We found that mutation at the nucleophilic residue (D200) did not provide a typical glycosynthase from this bacterial enzyme, but several mutants with mutation at the general acid/base residue E274 of the Lactobacillus casei α1,6-fucosidase, including E274A, E274S, and E274G, acted as efficient glycoligases that could fucosylate a wide variety of complex N-glycopeptides and intact glycoproteins by using α-fucosyl fluoride as a simple donor substrate. Studies on the substrate specificity revealed that the α1,6-fucosidase mutants could introduce an α1,6-fucose moiety specifically at the Asn-linked GlcNAc moiety not only to GlcNAc-peptide, but also to high-mannose and complex type N-glycans in the context of N-glycopeptides, N-glycoproteins, and intact antibodies. This discovery opens a new avenue to a wide variety of homogeneous, core-fucosylated N-glycopeptides and N-glycoproteins that are hitherto difficult to obtain for structural and functional studies.

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Section: Resultsmentioning

confidence: 99%

Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

Zhu

Christopher

et al. 2017

J. Am. Chem. Soc.

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“…Selection and evolution of those glycosidases with inherent transglycosylation activity could be a good starting point to generate new glycoligases and glycosynthases as well. 170,174,176 The development of novel glycosynthase and glycoligases provides exciting new tools for oligosaccharide and glycoprotein synthesis.…”

Section: Glycosidase-catalyzed Transglycosylation and The Glycosynthamentioning

confidence: 99%

Chemoenzymatic Methods for the Synthesis of Glycoproteins

2018

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Glycosylation is one of the most prevalent posttranslational modifications that profoundly affects the structure and functions of proteins in a wide variety of biological recognition events. However, the structural complexity and heterogeneity of glycoproteins, usually resulting from the variations of glycan components and/or the sites of glycosylation, often complicates detailed structure-function relationship studies and hampers the therapeutic applications of glycoproteins. To address these challenges, various chemical and biological strategies have been developed for producing glycan-defined homogeneous glycoproteins. This review highlights recent advances in the development of chemoenzymatic methods for synthesizing homogeneous glycoproteins, including the generation of various glycosynthases for synthetic purposes, endoglycosidase-catalyzed glycoprotein synthesis and glycan remodeling, and direct enzymatic glycosylation of polypeptides and proteins. The scope, limitation, and future directions of each method are discussed.

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“…In this regard, it should be noticed that, apart from its broad acceptor promiscuity, the preferred acidic pH range for rBxTW1-E495A from 2.2 to 5.5 has the additional advantage of enhancing the stability of both the donor and some products. Namely, xylosyl esters and xylosyl phosphoesters are sensitive to neutral pH, therefore even in the case that one of the few known examples of thioglycoligases forming C-O-C linkages 23,70 was able to use a phosphoric or a carboxylic acid acceptor, the production of the corresponding sugar ester would be compromised by their neutral or alkaline reaction pH (Supplementary Table 1).…”

Section: Conversion Of Rbxtw1 Into a Thioglycoligasementioning

confidence: 99%

“…More recently, several phenols 18 and an extended set of carboxylic acids 19 have been tested as acceptors, expanding the scope of the reaction, which, even so, is still limited. In spite of the large repertoire of glycosidases classified in more than 160 families to date (CAZy data base), research on thioglycoligases has involved just a few enzymes from GH families 1,2,11,13,20,31,35,51, and 89 [20][21][22][23][24][25][26][27][28][29] , most of which display maximal activity in a pH range from 6 to 8 and are unable to function under more acidic conditions (Supplementary Table 1).…”

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confidence: 99%

Thioglycoligase derived from fungal GH3 β-xylosidase is a multi-glycoligase with broad acceptor tolerance

et al. 2020

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The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal β-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the pKa of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis.

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Transglycosylation of engineered cyclodextrin glucanotransferases as O-glycoligases

Cited by 14 publications

References 36 publications

Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

Chemoenzymatic Methods for the Synthesis of Glycoproteins

Thioglycoligase derived from fungal GH3 β-xylosidase is a multi-glycoligase with broad acceptor tolerance

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