Title: N-glycosylation affects substrate specificity of chicory fructan 1-exohydrolase: evidence for the presence of an inulin binding cleft
Authors: Le Roy, Katrien ×
Verhaest, Maureen
Rabijns, Anja
Clerens, Stefan
Van Laere, André
Van den Ende, Wim #
Issue Date: 2007
Publisher: Wiley-Blackwell Publishing Ltd.
Series Title: New Phytologist vol:176 issue:2 pages:317-324
Abstract: * Recently, the three-dimensional structure of chicory (Cichorium intybus) fructan 1-exohydrolase (1-FEH IIa) in complex with its preferential substrate, 1-kestose, was determined. Unfortunately, no such data could be generated with high degree of polymerization (DP) inulin, despite several soaking and cocrystallization attempts. * Here, site-directed mutagenesis data are presented, supporting the presence of an inulin-binding cleft between the N- and C-terminal domains of 1-FEH IIa. In general, enzymes that are unable to degrade high DP inulins contain an N-glycosylation site probably blocking the cleft. By contrast, inulin-degrading enzymes have an open cleft configuration. * An 1-FEH IIa P294N mutant, introducing an N-glycosylation site near the cleft, showed highly decreased activity against higher DP inulin. The introduction of a glycosyl chain most probably blocks the cleft and prevents inulin binding and degradation. * Besides cell wall invertases, fructan 6-exohydrolases (6-FEHs) also contain a glycosyl chain most probably blocking the cleft. Removal of this glycosyl chain by site-directed mutagenesis in Arabidopsis thaliana cell wall invertase 1 and Beta vulgaris 6-FEH resulted in a strong decrease of enzymatic activities of the mutant proteins. By analogy, glycosylation of 1-FEH IIa affected overall enzyme activity. These data strongly suggest that the presence or absence of a glycosyl chain in the cleft is important for the enzyme's stability and optimal conformation.
ISSN: 0028-646X
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Molecular Physiology of Plants and Micro-organisms Section - miscellaneous
Faculty of Pharmaceutical Sciences - miscellaneous
Centre for Food and Microbial Technology
Research Group Neuroplasticity and Neuroproteomics (-)
× corresponding author
# (joint) last author

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