Acceptor-dependent regioselectivity of glycosynthase reactions by Streptomyces E383A β-glucosidase

Faijes, Magda; Saura-Valls, Marc; Pérez, Xavi; Conti, Marta Helena Souza De; Planas, Antoni

doi:10.1016/j.carres.2006.04.049

Cited by 32 publications

(23 citation statements)

References 27 publications

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“…This result opened a new route for carbohydrate synthesis, and already several retaining glycosidases have been successfully converted to glycosynthases with this strategy. [12][13][14][15][16][17][18][19][20][21][22][23] Directed evolution would offer an obvious route to improve the activity and alter the substrate selectivity of these enzymes, except that there is no intrinsic way to screen or select for glycosynthase activity. Mayer et al developed a coupled enzyme assay using an endo-cellullase that can be used to screen for glycosynthase mutants with improved activity.…”

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confidence: 99%

Characterization of a New Glycosynthase Cloned by Using Chemical Complementation

Tao¹,

Peralta‐Yahya²,

Decatur³

et al. 2008

ChemBioChem

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mentioning

confidence: 99%

Characterization of a New Glycosynthase Cloned by Using Chemical Complementation

Tao¹,

Peralta‐Yahya²,

Decatur³

et al. 2008

ChemBioChem

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“…[4][5][6][7][8][9] Exoglycosynthases, glycosynthases derived from exoglycosidases, have moderate substrate specificity and regioselectivity and can synthesize short chain oligosaccharides (di-, tri-, and tetra-oligosaccharides) that have various glycosidic linkages. [10][11][12] Endoglycosynthases, which are derived from endoglycosidases, generally have high regioselectivity and can catalyze the synthesis of specific glycosidic linkages. They can synthesize longer oligosaccharides than exoglycosynthases because they have long glyconebinding sites that accommodate longer glycosyl donor substrates.…”

Section: Introductionmentioning

confidence: 99%

The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase

et al. 2012

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Rice BGlu1 b-glucosidase nucleophile mutant E386G is a glycosynthase that can synthesize p-nitrophenyl (pNP)-cellooligosaccharides of up to 11 residues. The X-ray crystal structures of the E386G glycosynthase with and without a-glucosyl fluoride were solved and the a-glucosyl fluoride complex was found to contain an ordered water molecule near the position of the nucleophile of the BGlu1 native structure, which is likely to stabilize the departing fluoride. The structures of E386G glycosynthase in complexes with cellotetraose and cellopentaose confirmed that the side chains of N245, S334, and Y341 interact with glucosyl residues in cellooligosaccharide binding subsites 12, 13, and 14. Mutants in which these residues were replaced in BGlu1 b-glucosidase hydrolyzed cellotetraose and cellopentaose with k cat /K m values similar to those of the wild type enzyme. However, the Y341A, Y341L, and N245V mutants of the E386G glycosynthase synthesize shorter pNPcellooligosaccharides than do the E386G glycosynthase and its S334A mutant, suggesting that Y341 and N245 play important roles in the synthesis of long oligosaccharides. X-ray structural studies revealed that cellotetraose binds to the Y341A mutant of the glycosynthase in a very different, alternative mode not seen in complexes with the E386G glycosynthase, possibly explaining the similar hydrolysis, but poorer synthesis of longer oligosaccharides by Y341 mutants.

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“…The use of these enzymes for making glycosides has become an attractive alternative to chemical synthesis for the perfect control of the regio-and stereochemistry of glycosylation. Different enzymatic methodologies using glycosidases and glycosynthases (Faijes et al 2006;Hancock et al 2006;Murata & Usui 2006;Perugino et al 2005), as well as glycosyltransferases (Blixt & Razi 2006;Hanson et al 2004) have been successfully used in the synthetic direction.…”

Section: Biocatalytic Glycosyl Transfer Reactionsmentioning

confidence: 99%

Tools and ingredients for the biocatalytic synthesis of carbohydrates and glycoconjugates

Wohlgemuth¹

2008

Biocatalysis and Biotransformation

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The presence of multiple functional groups and stereocentres in carbohydrates and glycoconjugates make them challenging targets for synthesis. Although progress in chemical synthesis and engineering is impressive, there is still a need to selectively introduce and remove protecting groups in the total synthesis of target molecules of increasing complexity. Multiple hydroxyl-groups with similar reactivities have to be differentiated in order to form the desired glycosidic bonds in a regioand stereospecific way. To complement the existing chemical tools and ingredients, biocatalysts for selective carbonÁcarbon bond formation and glycosylation reactions have been developed. The availability of auxiliary ingredients like transfer reagents is a prerequisite for the development of viable biocatalytic process steps. In the case of dihydroxyacetonephosphate-dependent aldolases, e.g. fructose-1,6-bisphosphate aldolase (EC 4.1.2.13), the large-scale availability of dihydroxyacetone-phosphate (DHAP) eliminates the need to synthesize the donor DHAP. For the pyruvate-dependent aldolases, e.g. the N-acetylneuraminic acid aldolase (EC 4.1.3.3) and acetaldehyde-dependent aldolases like the 2-deoxy-Dribose-5-phosphate aldolase (4.2.1.4), the donors pyruvate and acetaldehyde are also available on a large scale. A broad range of natural and recombinant aldolases have been produced in stable lyophilized form. Recombinant transketolase together with a new synthesis of hydroxypyruvates has provided a platform technology for the preparation of monosaccharides, whereby the carbon backbone is extended by a two-carbon unit (C2-elongation). Natural and recombinant glycosyltransferases have been prepared on a large-scale to establish biocatalytic glycosylations in water as highly regio-and stereospecific reaction methodologies without the need for laborious protecting group manipulations, solubility adaptations and complex synthetic schemes. In order to simplify the synthetic manipulations for specific glycosylations, toolkits for b-1,4-galactosylations, a-1,3-galactosylations and a-1,3-fucosylations have been developed for rapid quantitative conversions. The introduction of matched pairs of biocatalysts and transfer reagents as ingredients together with the optimized reaction methodology as tool provide an important starting point for biocatalytic glycomics.

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Acceptor-dependent regioselectivity of glycosynthase reactions by Streptomyces E383A β-glucosidase

Cited by 32 publications

References 27 publications

Characterization of a New Glycosynthase Cloned by Using Chemical Complementation

Characterization of a New Glycosynthase Cloned by Using Chemical Complementation

The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase

Tools and ingredients for the biocatalytic synthesis of carbohydrates and glycoconjugates

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