Strategy for Converting an Inverting Glycoside Hydrolase into a Glycosynthase

Motomitsu Kitaoka, Yuji Honda, Masafumi Hidaka, Shinya Fushinobu

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Citations (Scopus)

Abstract

We found a novel inverting xylanolytic enzyme belonging to GH8, reducing end xylose-releasing exo-oligoxylanase (Rex, EC. 3.2.1.156), that hydrolyzed xylooligosaccharides (X3 or larger) to release X1 at their reducing end. Rex hydrolyzed α-X2F into X2 only in the presence of X1, clearly proving the Hehre-resynthesis hydrolysis mechanism. A library of mutant Rex at the catalytic base (Asp263) was constructed by saturation mutagenesis. Among them, D263C showed the highest level of X3 production, and D263N exhibited the fastest consumption of α-X2F. However, F- releasing activities of the mutants were much less than that of wild type. Next, Y198 of Rex that forms a hydrogen bond with the nucleophilic water was substituted with phenylalanine, causing a drastic decrease in the hydrolytic activity and a small increase in F- releasing activity from α-xylobiosyl fluoride in the presence of xylose. Y198F of Rex accumulates much more product during the glycosynthase reaction than D263C. We here conclude that an inverting glycosidase is effectively converted into glycosynthase by mutating a residue holding the nucleophilic water molecule with the general base residue while keeping the general base residue intact.

Original languageEnglish
Title of host publicationCarbohydrate-Active Enzymes
Subtitle of host publicationStructure, Function and Applications
PublisherElsevier Ltd
Pages193-205
Number of pages13
ISBN (Print)9781845695194
DOIs
Publication statusPublished - 2008 Sep
Externally publishedYes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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    Kitaoka, M., Honda, Y., Hidaka, M., & Fushinobu, S. (2008). Strategy for Converting an Inverting Glycoside Hydrolase into a Glycosynthase. In Carbohydrate-Active Enzymes: Structure, Function and Applications (pp. 193-205). Elsevier Ltd. https://doi.org/10.1533/9781845695750.2.193