Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Plant Sciences, fructan 1-exohydrolase (1-FEH IIa), invertase, N-glycosylation, site-directed mutagenesis, substrate binding cleft, substrate specificity, CELL-WALL INVERTASE, X-RAY-DIFFRACTION, FUNCTIONAL-ANALYSIS, ACTIVE-SITE, METABOLIZING ENZYMES, BETA-FRUCTOSIDASE, CRYSTAL-STRUCTURE, MASS FINGERPRINT, CLONING, WHEAT, Amino Acid Sequence, Arabidopsis, Arabidopsis Proteins, Beta vulgaris, Binding Sites, Cell Wall, Cichorium intybus, Glycoside Hydrolases, Glycosylation, Inulin, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Plant Proteins, Protein Structure, Tertiary, Sequence Alignment, Substrate Specificity, beta-Fructofuranosidase, 06 Biological Sciences, 07 Agricultural and Veterinary Sciences, Plant Biology & Botany, 3108 Plant biology, 4101 Climate change impacts and adaptation, 4102 Ecological applications

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.