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Efficient glycosynthase mutant derived from Mucor hiemalis endo-beta-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan.


ABSTRACT: Endo-M, an endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is a family 85 glycoside hydrolase. This enzyme is unique in that it can transfer en bloc the oligosaccharide of various types of N-glycans onto different acceptors, and thereby it enzymatically generates diverse glycoconjugates. In this study, we performed mutational and kinetic studies focusing on a key catalytic asparagine 175 of Endo-M. We have shown that most of the Asn-175 mutants had significantly diminished hydrolysis activity but acted as glycosynthases capable of using synthetic sugar oxazoline for transglycosylation. Our results confirm the critical role of this asparagine residue in promoting the formation of an oxazolinium ion intermediate in the first step of the substrate-assisted catalysis. Interestingly, the N175Q mutant was found to possess dramatically enhanced glycosynthase-like activity with sugar oxazoline in comparison with N175A and a transglycosidase-like activity with "natural" N-glycan as well. These results also implicated the significance of amide side chain in the asparagine 175 of Endo-M for promoting oxazoline transglycosylation in the second step of the catalysis. The highly efficient syntheses of glycopeptides/glycoproteins by N175Q combined with synthetic sugar oxazolines or natural N-glycan substrates were exemplified. In addition, we also identified several previously unknown residues that seem to play a role in the catalysis of Endo-M.

SUBMITTER: Umekawa M 

PROVIDER: S-EPMC2804199 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Publications

Management of central venous catheters in pediatric onco-hematology using 0.9% sodium chloride and positive-pressure-valve needleless connector.

Buchini Sara S   Scarsini Sara S   Montico Marcella M   Buzzetti Roberto R   Ronfani Luca L   Decorti Cinzia C  

European journal of oncology nursing : the official journal of European Oncology Nursing Society 20140413 4


<h4>Purpose</h4>To describe, in a sample of pediatric onco-hematological patients, the rate of occlusions in unused central venous catheters (CVC) flushed once a week with a 0.9% sodium chloride solution through a positive-pressure-valve needleless connector.<h4>Method</h4>Retrospective cohort study. Subjects aged 0-17 years were identified through a manual search in medical and nursing records and were observed for two years or until the occurrence of one of the following events: start or resum  ...[more]