Glycosynthase with Broad Substrate Specificity – an Efficient Biocatalyst for the Construction of Oligosaccharide Library

A versatile glycosynthase (TnG-E338A) with strikingly broad substrate scope has been developed from Thermus nonproteolyticus β-glycosidase (TnG) by using site-directed mutagenesis. The practical utility of this biocatalyst has been demonstrated by the facile generation of a small library containing various oligosaccharides and a steroidal glycoside (total 25 compounds) in up to 100 % isolated yield. Moreover, an array of eight gluco-oligosaccharides has been readily synthesized by the enzyme in a one-pot, parallel reaction, which highlights its potential in the combinatorial construction of a carbohydrate library that will assist glycomic and glycotherapeutic research. Significantly, the enzyme provides a means by which glycosynthase technology may be extended to combinatorial chemistry.

[1]  S. Withers,et al.  Rice BGlu1 glycosynthase and wild type transglycosylation activities distinguished by cyclophellitol inhibition. , 2012, Carbohydrate research.

[2]  J. Thorson,et al.  Enzymatic methods for glyco(diversification/randomization) of drugs and small molecules. , 2011, Natural product reports.

[3]  S. Withers,et al.  Glycosphingolipid synthesis employing a combination of recombinant glycosyltransferases and an endoglycoceramidase glycosynthase. , 2011, Chemical communications.

[4]  Lothar Elling,et al.  Combinatorial One‐Pot Synthesis of Poly‐N‐acetyllactosamine Oligosaccharides with Leloir‐Glycosyltransferases , 2011 .

[5]  M. Rossi,et al.  Glycosynthases in Biocatalysis , 2011 .

[6]  Lai-Xi Wang,et al.  Convergent synthesis of homogeneous Glc1Man9GlcNAc2-protein and derivatives as ligands of molecular chaperones in protein quality control. , 2011, Journal of the American Chemical Society.

[7]  H. Brumer,et al.  Building Custom Polysaccharides in Vitro with an Efficient, Broad-Specificity Xyloglucan Glycosynthase and a Fucosyltransferase , 2011, Journal of the American Chemical Society.

[8]  S. Withers,et al.  Toward Efficient Enzymatic Glycan Synthesis: Directed Evolution and Enzyme Engineering , 2011 .

[9]  Shane M. Wilkinson,et al.  Experimental and kinetic studies of the Escherichia coli glucuronylsynthase: an engineered enzyme for the synthesis of glucuronide conjugates. , 2011, The Journal of organic chemistry.

[10]  S. Withers,et al.  New approaches to enzymatic glycoside synthesis through directed evolution. , 2010, Carbohydrate research.

[11]  Masahiro Samejima,et al.  Synthesis of cyclic beta-glucan using laminarinase 16A glycosynthase mutant from the basidiomycete Phanerochaete chrysosporium. , 2010, Journal of the American Chemical Society.

[12]  Thomas J Boltje,et al.  Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research. , 2009, Nature chemistry.

[13]  M. Rossi,et al.  beta-Glycosyl azides as substrates for alpha-glycosynthases: preparation of efficient alpha-L-fucosynthases. , 2009, Chemistry & biology.

[14]  Beat Ernst,et al.  From carbohydrate leads to glycomimetic drugs , 2009, Nature Reviews Drug Discovery.

[15]  S. Withers,et al.  Designer enzymes for glycosphingolipid synthesis by directed evolution. , 2009, Nature chemical biology.

[16]  Wei Zhang,et al.  Discovery of the first series of small molecule H5N1 entry inhibitors. , 2009, Journal of medicinal chemistry.

[17]  V. Křen,et al.  Glycosidases: a key to tailored carbohydrates. , 2009, Trends in biotechnology.

[18]  Shane M. Wilkinson,et al.  Escherichia coli glucuronylsynthase: an engineered enzyme for the synthesis of beta-glucuronides. , 2008, Organic letters.

[19]  Carolyn R Bertozzi,et al.  Synthetic glycobiology: Exploits in the Golgi compartment. , 2006, Current opinion in chemical biology.

[20]  S. Withers,et al.  Engineering of glycosidases and glycosyltransferases. , 2006, Current opinion in chemical biology.

[21]  J. Thorson,et al.  Exploiting the Reversibility of Natural Product Glycosyltransferase-Catalyzed Reactions , 2006, Science.

[22]  S. Withers,et al.  Glycosynthase-mediated synthesis of glycosphingolipids. , 2006, Journal of the American Chemical Society.

[23]  S. Withers,et al.  Enzymatic transglycosylation of xylose using a glycosynthase. , 2005, Carbohydrate research.

[24]  M. Dion,et al.  Thermus thermophilus Glycosynthases for the Efficient Synthesis of Galactosyl and Glucosyl β‐(1→3)‐Glycosides , 2005 .

[25]  Hening Lin,et al.  Directed evolution of a glycosynthase via chemical complementation. , 2004, Journal of the American Chemical Society.

[26]  S. Withers,et al.  Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire* , 2004, Journal of Biological Chemistry.

[27]  G. Haki,et al.  Developments in industrially important thermostable enzymes: a review. , 2003, Bioresource technology.

[28]  Xinquan Wang,et al.  Structural Basis for Thermostability of β-Glycosidase from the Thermophilic Eubacterium Thermus nonproteolyticus HG102 , 2003, Journal of bacteriology.

[29]  P. Seeberger,et al.  Combinatorial carbohydrate chemistry. , 2002, Current opinion in chemical biology.

[30]  Y. Shoujun,et al.  Cloning and expression of thermostable β-glycosidase gene from thermus nonproteolyticus HG102 and characterization of recombinant enzyme , 2001 .

[31]  S. Withers,et al.  β-Mannosynthase: Synthesis ofβ-Mannosides with a Mutantβ-Mannosidase , 2001 .

[32]  S. Withers,et al.  Glycosyl fluorides in enzymatic reactions. , 2000, Carbohydrate research.

[33]  L. Christiansen,et al.  Highly efficient synthesis of β(1, 4)-oligo- and polysaccharides using a mutant cellulase , 2000 .

[34]  M. Rossi,et al.  A novel thermophilic glycosynthase that effects branching glycosylation. , 2000, Bioorganic & medicinal chemistry letters.

[35]  S. Withers,et al.  The E358S mutant of Agrobacterium sp. β‐glucosidase is a greatly improved glycosynthase , 2000, FEBS letters.

[36]  S. Withers,et al.  Glycosynthases: Mutant Glycosidases for Oligosaccharide Synthesis , 1998 .

[37]  B Henrissat,et al.  Structural and sequence-based classification of glycoside hydrolases. , 1997, Current opinion in structural biology.

[38]  V. Vetvicka,et al.  Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. , 1996, The Journal of clinical investigation.

[39]  Raymond A. Dwek,et al.  Glycobiology: Toward Understanding the Function of Sugars. , 1996, Chemical reviews.

[40]  G. Look,et al.  Enzyme-catalyzed oligosaccharide synthesis. , 1992, Analytical biochemistry.

[41]  P. Garegg.  Thioglycosides as glycosyl donors in oligosaccharide synthesis. , 1997, Advances in carbohydrate chemistry and biochemistry.