Anatomy of glycosynthesis: Structure and kinetics of the Humicola insolens Cel7B E197A and E197S glycosynthase mutants

VMA Ducros, CA Tarling, DL Zechel, AM Brzozowski, TP Frandsen, Ingemar von Ossowski, M Schulein, SG Withers, GJ Davies*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

Abstract

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).

Original languageEnglish
Pages (from-to)619-628
Number of pages10
JournalCHEMISTRY AND BIOLOGY
Volume10
Issue number7
DOIs
Publication statusPublished - Jul 2003
MoE publication typeA1 Journal article-refereed

Keywords

  • CATALYTIC NUCLEOPHILE
  • BETA-GLUCOSIDASE
  • ENDOGLUCANASE I
  • FUSARIUM-OXYSPORUM
  • ACTIVE-SITE
  • IDENTIFICATION
  • GLYCOSIDASES
  • GALACTOSIDASES
  • GLYCOSYLATION
  • SPECIFICITY

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