N-glycosylation affects substrate specificity of chicory fructan 1-exohydrolase: evidence for the presence of an inulin binding cleft
Article first published online: 26 JUL 2007
© The Authors (2007). Journal compilation © New Phytologist (2007)
Volume 176, Issue 2, pages 317–324, October 2007
How to Cite
Le Roy, K., Verhaest, M., Rabijns, A., Clerens, S., Van Laere, A. and Van den Ende, W. (2007), N-glycosylation affects substrate specificity of chicory fructan 1-exohydrolase: evidence for the presence of an inulin binding cleft. New Phytologist, 176: 317–324. doi: 10.1111/j.1469-8137.2007.02174.x
- Issue published online: 26 JUL 2007
- Article first published online: 26 JUL 2007
- Received: 21 March 2007 Accepted: 30 May 2007
- fructan 1-exohydrolase (1-FEH IIa);
- site-directed mutagenesis;
- substrate binding cleft;
- substrate specificity
- • 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.