1ojk: Difference between revisions
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==Overview== | ==Overview== | ||
The formation of glycoconjugates and oligosaccharides remains one of the | The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat). | ||
==About this Structure== | ==About this Structure== | ||
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[[Category: Humicola insolens]] | [[Category: Humicola insolens]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Brzozowski, A | [[Category: Brzozowski, A M.]] | ||
[[Category: Davies, G | [[Category: Davies, G J.]] | ||
[[Category: Ducros, V | [[Category: Ducros, V M.A.]] | ||
[[Category: Frandsen, T | [[Category: Frandsen, T P.]] | ||
[[Category: Ossowski, I | [[Category: Ossowski, I Von.]] | ||
[[Category: Schulein, M.]] | [[Category: Schulein, M.]] | ||
[[Category: Tarling, C | [[Category: Tarling, C A.]] | ||
[[Category: Withers, S | [[Category: Withers, S G.]] | ||
[[Category: Zechel, D | [[Category: Zechel, D L.]] | ||
[[Category: GOL]] | [[Category: GOL]] | ||
[[Category: NAG]] | [[Category: NAG]] | ||
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[[Category: hydrolase]] | [[Category: hydrolase]] | ||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:18:27 2008'' | ||
Revision as of 15:18, 21 February 2008
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ANATOMY OF GLYCOSYNTHESIS: STRUCTURE AND KINETICS OF THE HUMICOLA INSOLENS CEL7BE197A AND E197S GLYCOSYNTHASE MUTANTS
OverviewOverview
The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).
About this StructureAbout this Structure
1OJK is a Single protein structure of sequence from Humicola insolens with and as ligands. Active as Cellulase, with EC number 3.2.1.4 Known structural/functional Site: . Full crystallographic information is available from OCA.
ReferenceReference
Anatomy of glycosynthesis: structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants., Ducros VM, Tarling CA, Zechel DL, Brzozowski AM, Frandsen TP, von Ossowski I, Schulein M, Withers SG, Davies GJ, Chem Biol. 2003 Jul;10(7):619-28. PMID:12890535
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