title: N-glycosylation affects substrate specificity of chicory fructan 1-exohydrolase: evidence for the presence of an inulin binding cleft.
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.
authors: K. U. Leuven, Laboratory of Molecular Plant Physiology, Institute of Botany and Microbiology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium.
authors: Katrien
authors: K
authors: Le Roy
authors: K. U. Leuven, Laboratory of Biocrystallography, Herestraat 49, O & N II box 822, B-3000 Leuven, Belgium.
authors: Maureen
authors: M
authors: Verhaest
authors: K. U. Leuven, Laboratory of Biocrystallography, Herestraat 49, O & N II box 822, B-3000 Leuven, Belgium.
authors: Anja
authors: A
authors: Rabijns
authors: K. U. Leuven, Laboratory of Neuroplasticity and Neuroproteomics, Naamsestraat 59, B-3000 Leuven, Belgium.
authors: Stefan
authors: S
authors: Clerens
authors: K. U. Leuven, Laboratory of Molecular Plant Physiology, Institute of Botany and Microbiology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium.
authors: André
authors: A
authors: Van Laere
authors: K. U. Leuven, Laboratory of Molecular Plant Physiology, Institute of Botany and Microbiology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium.
authors: Wim
authors: W
authors: Van den Ende
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D027461:Beta vulgaris / Q000201:enzymology; D001665:Binding Sites; D002473:Cell Wall / Q000201:enzymology; D018651:Cichorium intybus / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D007444:Inulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013379:Substrate Specificity; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D010940:Plant Proteins; D007444:Inulin; D006026:Glycoside Hydrolases; C517207:Inv1 protein, Arabidopsis; D043324:beta-Fructofuranosidase; C044118:fructan beta-fructosidase