Simultaneous detection of different glycosidase activities by 19F NMR spectroscopy

Background: Catalytic residues and molecular mechanism of GH-66 enzymes were hitherto unknown. Results: Novel dextranase produced isomaltotetraose and cyclo-isomaltosaccharides. Its nucleophile (Asp 340 ) and acid/base catalyst (Glu 412 ) were identified by a chemical rescue approach. Conclusion: Three GH-66 enzyme types were newly classified for the first time. Significance: This work elucidates production of isomaltotetraose and cycloisomaltosaccharides, classification of GH-66, identification of catalytic residues, and novel dextran-forming type chemical rescue.

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“…Purified IG-4 (100 mg) was converted to ␣-isomaltotetraosyl fluoride (IG4F) (24) for the substrate used in the ChR reaction.…”

Section: Methodsmentioning

confidence: 99%

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

Kim

Kiso

Muraki

et al. 2012

Journal of Biological Chemistry

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“…Fucose liberated in the absence of Lac or enzyme could be a product of the spontaneous hydrolysis of FucF because the degradation was observed to a similar extent when FucF alone was incubated in the buffer (lane 3). It is well known that β‐glycosyl fluorides are unstable in aqueous solution [8,18]. Hehre resynthesis‐hydrolysis was demonstrated for the wild‐type Fuc domain.…”

Section: Resultsmentioning

confidence: 99%

1,2‐α‐l‐Fucosynthase: A glycosynthase derived from an inverting α‐glycosidase with an unusual reaction mechanism

et al. 2008

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Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fuca1,2-Gal linkage by introducing glycosynthase technology into 1,2-a-L L-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using b-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2 0 -fucosyllactose. 1,2-a-L L-Fucosynthase is the first glycosynthase derived from an inverting a-glycosidase and from a glycosidase with an unusual reaction mechanism.

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“…First, β -glycosyl-F ( equatorial bonds) have much shorter half-lives than α -ones ( axial bonds) because of the anomeric effect (Albert et al 2000;Juaristi and Cuevas 1992). Thus, βglycosyl-F might be too unstable to resist the long reaction times necessary for α -glycosynthase reactions.…”

Section: β -Glycosyl Azides As New Substrates For α -Glycosynthasesmentioning

confidence: 99%

Exploitation of β-glycosyl azides for the preparation of α-glycosynthases

Cobucci‐Ponzano

Zorzetti

Strazzulli

et al. 2012

Biocatalysis and Biotransformation

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An interesting approach for the chemo-enzymatic synthesis of carbohydrates is the use of glycosynthases, a class of mutant glycosidases derived from β -glycoside hydrolases obtained by replacement of the active site nucleophile with a non-nucleophilic residue. However, the scarcity of α -glycosynthases has so far hampered access to the synthesis of a large class of oligosaccharides of biotechnological interest. We review here a new glycosynthetic methodology for the production of two retaining α -fucosynthases and an α -galactosynthase exploiting β -glycosyl azide derivatives. The general applicability of this approach, which opens new perspectives in the use of azide derivatives for the production of novel α -glycosynthases, is discussed.

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Simultaneous detection of different glycosidase activities by 19F NMR spectroscopy

Cited by 22 publications

References 14 publications

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

1,2‐α‐l‐Fucosynthase: A glycosynthase derived from an inverting α‐glycosidase with an unusual reaction mechanism

Exploitation of β-glycosyl azides for the preparation of α-glycosynthases

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