N‐glycosylation affects substrate specificity of chicory fructan 1‐exohydrolase: evidence for the presence of an inulin binding cleft

Abstract

Summary• 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 sitedirected 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.

Towards understanding vacuolar antioxidant mechanisms: a role for fructans?

“…S4 at JXB online). Additional evidence was generated with enzyme reactions containing either chicory 1-FEH (fructan-1-exohydrolase) IIa (Le Roy et al ., 2007) or sugar beet 6-FEH (Van den Ende et al ., 2003) (Fig. 3).…”

Water stress drastically reduces root growth and inulin yield in Cichorium intybus (var. sativum) independently of photosynthesis

“…Kusch et al (2009) recently demonstrated that numerous biochemical factors (including Ca 2+ signalling, protein kinases, and phosphatases) are directly involved in the modulation of the expression of the gene encoding 1-FFT. Similarly, the FEH enzyme activity depends on three distinct isoforms (1-FEHI, IIa, and IIb) that may exhibit various patterns of regulation (Van Laere and Van den Ende, 2002; Van den Ende et al, 2004; Le Roy et al, 2007; Kusch et al, 2009). …”

Mapping the N-linked glycosites of rice (Oryza sativa L.) germinating embryos

“…For the A . thaliana cell wall invertase 1-FEH IIa, N -glycosylation has been shown to be critical for maintaining both enzyme stability and its optimal conformation [52]. We therefore hypothesized that glycosylation modification is involved in the sucrose metabolism possibly by regulating the enzyme activity of key regulators such as CIN2/GIF1.…”