Teaching old enzymes new tricks: engineering and evolution of glycosidases and glycosyl transferases for improved glycoside synthesis.

The therapeutic potential of glycosides has made them an attractive target for drug development. The biological extraction and chemical synthesis of these molecules is often challenging and low yielding, thus alternative methods for the synthesis of polysaccharides are being pursued. A new class of enzymes, glycosynthases, which are nucleophile mutants of glycosidases, can perform the transglycosylation reaction without hydrolyzing the product, and thus provide a valuable resource for polysaccharide and glycan synthesis. Directed evolution of glycosynthases has expanded the repertoire of glycosidic linkages formed and the donors and acceptors (both sugar and nonsugar) that can be used by the glycosynthase. The application of new screening methods, such as FACS, to the directed evolution of glycosynthases will aid in the development of enzymes that are able to efficiently synthesize new, and therapeutically relevant glycosidic linkages.

[1]  S. Hakomori.  Cell adhesion/recognition and signal transduction through glycosphingolipid microdomain , 2000, Glycoconjugate Journal.

[2]  S. Withers,et al.  Synthesis with Glycosynthases: Cello-Oligomers of Isofagomine and a Tetrahydrooxazine as Cellulase Inhibitors , 2002 .

[3]  B. Saha,et al.  Hemicellulose bioconversion , 2003, Journal of Industrial Microbiology and Biotechnology.

[4]  S. Withers,et al.  Dissection of nucleophilic and acid-base catalysis in glycosidases. , 2001, Current opinion in chemical biology.

[5]  S. Withers,et al.  Mechanistic consequences of mutation of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase. , 1992, Biochemistry.

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

[7]  G. Tettamanti.  Ganglioside/glycosphingolipid turnover: New concepts , 2003, Glycoconjugate Journal.

[8]  H. Yamana,et al.  Glycotherapy for cancer: remodeling of ganglioside pattern as an effective approach for cancer therapy. , 2002, Cancer detection and prevention.

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

[10]  Specificity of carbohydrate structures of gangliosides in the activity to regenerate the rat axotomized hypoglossal nerve. , 2001, Glycobiology.

[11]  J. B. Kempton,et al.  Mechanism of Agrobacterium beta-glucosidase: kinetic studies. , 1992, Biochemistry.

[12]  Chi‐Huey Wong,et al.  Chemoenzymatic synthesis of oligosaccharides and glycoproteins. , 2004, Trends in biochemical sciences.

[13]  Chi‐Huey Wong,et al.  Chemoenzymatic Synthesis of Oligosaccharides and Glycoproteins , 2005 .

[14]  G. Bråne,et al.  Alzheimer Disease – Effect of Continuous Intracerebroventricular Treatment with GM1 Ganglioside and a Systematic Activation Programme , 2002, Dementia and Geriatric Cognitive Disorders.

[15]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

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

[17]  V. Cervelli,et al.  Gangliosides potentiate the immunosuppressive effects of cyclosporin A in rat skin allografts. , 1992, Transplant international : official journal of the European Society for Organ Transplantation.

[18]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[19]  S. Withers,et al.  Mechanisms of enzymatic glycoside hydrolysis. , 1994, Current opinion in structural biology.

[20]  M. Kitaoka,et al.  The First Glycosynthase Derived from an Inverting Glycoside Hydrolase* , 2006, Journal of Biological Chemistry.

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

[22]  J Thiem.  Applications of enzymes in synthetic carbohydrate chemistry. , 1995, FEMS microbiology reviews.

[23]  L. Christiansen,et al.  Highly Efficient Synthesis of β(1→4)-Oligo- and Polysaccharides Using a Mutant Cellulase. , 2000 .

[24]  C. Bertozzi,et al.  Homogeneous glycopeptides and glycoproteins for biological investigation. , 2002, Annual review of biochemistry.

[25]  S. Withers,et al.  Glu280 is the nucleophile in the active site of Clostridium thermocellum CelC, a family A endo-beta-1,4-glucanase. , 1993, The Journal of biological chemistry.

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

[28]  S. Hakomori.  Glycosphingolipids in cellular interaction, differentiation, and oncogenesis. , 1981, Annual review of biochemistry.

[29]  S. Withers,et al.  Changing Enzymic Reaction Mechanisms by Mutagenesis: Conversion of a Retaining Glucosidase to an Inverting Enzyme , 1994 .

[30]  S. Hakomori.  Traveling for the glycosphingolipid path , 2000, Glycoconjugate Journal.

[31]  Spencer J. Williams,et al.  Glycosyl Fluorides in Enzymatic Reactions , 2000 .

[32]  Spencer J. Williams,et al.  Glycosynthases: Mutant Glycosidases for Glycoside Synthesis , 2002 .

[33]  A. Kondo,et al.  Molecular Cloning, Expression, and Sequence Analysis of the Endoglycoceramidase II Gene from Rhodococcus Species Strain M-777* , 1997, The Journal of Biological Chemistry.

[34]  S. Withers,et al.  Glycosynthase-based synthesis of xylo-oligosaccharides using an engineered retaining xylanase from Cellulomonas fimi. , 2006, Organic & biomolecular chemistry.

[35]  J. B. Kempton,et al.  Unequivocal demonstration of the involvement of a glutamate residue as a nucleophile in the mechanism of a retaining glycosidase , 1990 .

[36]  G. Davies,et al.  A glycosynthase catalyst for the synthesis of flavonoid glycosides. , 2007, Angewandte Chemie.

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

[38]  S. Withers,et al.  β-Mannosynthase: Synthesis of β-Mannosides with a Mutant β-Mannosidase. , 2001, Angewandte Chemie.

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

[40]  S. Withers,et al.  Directed evolution of new glycosynthases from Agrobacterium beta-glucosidase: a general screen to detect enzymes for oligosaccharide synthesis. , 2001, Chemistry & biology.

[41]  S. Withers,et al.  -Mannosynthase: Synthesis of -Mannosides with a Mutant -Mannosidase , 2001 .

[42]  Amir Aharoni,et al.  High-throughput screening methodology for the directed evolution of glycosyltransferases , 2006, Nature Methods.

[43]  H. Maccioni,et al.  Understanding the Stepwise Synthesis of Glycolipids , 2002, Neurochemical Research.

[44]  A. Pavan,et al.  Association of Cellular Prion Protein with Gangliosides in Plasma Membrane Microdomains of Neural and Lymphocytic Cells , 2002, Neurochemical Research.

[45]  S. Withers,et al.  Structural, Thermodynamic, and Kinetic Analyses of Tetrahydrooxazine-derived Inhibitors Bound to β-Glucosidases* , 2004, Journal of Biological Chemistry.

[46]  S. Withers,et al.  Structural and Mechanistic Analyses of endo-Glycoceramidase II, a Membrane-associated Family 5 Glycosidase in the Apo and GM3 Ganglioside-bound Forms* , 2007, Journal of Biological Chemistry.

[47]  S. Withers,et al.  Thermostable Glycosynthases and Thioglycoligases Derived from Thermotoga maritima β‐Glucuronidase , 2006, Chembiochem : a European journal of chemical biology.