N-glycosylation affects substrate specificity of chicory fructan 1-exohydrolase : evidence for the presence of an inulin binding cleft
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 an...
| Veröffentlicht in: | The New phytologist. - 1979. - 176(2007), 2 vom: 17., Seite 317-324 |
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| Weitere Verfasser: | , , , , |
| Format: | Online-Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2007
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| Zugriff auf das übergeordnete Werk: | The New phytologist |
| Schlagworte: | Journal Article Research Support, Non-U.S. Gov't Arabidopsis Proteins Plant Proteins Inulin 9005-80-5 Glycoside Hydrolases EC 3.2.1.- Inv1 protein, Arabidopsis EC 3.2.1.26 mehr... |
| Zusammenfassung: | 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 |
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| Beschreibung: | Date Completed 13.12.2007 Date Revised 09.01.2024 published: Print Citation Status MEDLINE |
| ISSN: | 1469-8137 |
| DOI: | 10.1111/j.1469-8137.2007.02174.x |