Insights into the fine architecture of the active site of chicory fructan 1-exohydrolase: 1-kestose as substrate vs sucrose as inhibitor
Article first published online: 11 JAN 2007
Volume 174, Issue 1, pages 90–100, April 2007
How to Cite
Verhaest, M., Lammens, W., Le Roy, K., De Ranter, C. J., Van Laere, A., Rabijns, A. and Van den Ende, W. (2007), Insights into the fine architecture of the active site of chicory fructan 1-exohydrolase: 1-kestose as substrate vs sucrose as inhibitor. New Phytologist, 174: 90–100. doi: 10.1111/j.1469-8137.2007.01988.x
- Issue published online: 26 JAN 2007
- Article first published online: 11 JAN 2007
- Received: 27 September 2006 Accepted: 24 November 2006
- fructan exohydrolase (FEH);
- glycoside hydrolase family 32;
- protein crystallization;
- X-ray crystallography
- • Invertases and fructan exohydrolases (FEHs) fulfil important physiological functions in plants. Sucrose is the typical substrate for invertases and bacterial levansucrases but not for plant FEHs, which are usually inhibited by sucrose.
- • Here we report on complexes between chicory (Cichorium intybus) 1-FEH IIa with the substrate 1-kestose and the inhibitors sucrose, fructose and 2,5 dideoxy-2,5-imino-D-mannitol. Comparisons with other family GH32 and 68 enzyme-substrate complexes revealed that sucrose can bind as a substrate (invertase/levansucrase) or as an inhibitor (1-FEH IIa).
- • Sucrose acts as inhibitor because the O2 of the glucose moiety forms an H-linkage with the acid-base catalyst E201, inhibiting catalysis. By contrast, the homologous O3 of the internal fructose in the substrate 1-kestose forms an intramolecular H-linkage and does not interfere with the catalytic process. Mutagenesis showed that W82 and S101 are important for binding sucrose as inhibitor.
- • The physiological implications of the essential differences in the active sites of FEHs and invertases/levansucrases are discussed. Sucrose-inhibited FEHs show a Ki (inhibition constant) well below physiological sucrose concentrations and could be rapidly activated under carbon deprivation.