A novel chicory fructanase can degrade common microbial fructan product profiles and displays positive cooperativity

Abstract

Fructan metabolism in bacteria and plants relies on fructosyltransferases and fructanases. Plant fructanases only hydrolyse terminal Fru residues (fructan exohydrolase, FEH). Levan (β-2,6 linkages) is the most abundant fructan type in bacteria. Dicot fructan accumulators, such as chicory (Cichorium intybus), accumulate inulin (β-2,1 linkages), harbouring several 1-FEH isoforms for their degradation. Here, a novel chicory fructanase with high affinity for levan was characterized, providing evidence that such enzymes widely occur in higher plants. It is adapted to common microbial fructan profiles, but has low affinity towards chicory inulin, in line with a function in trimming of microbial fructans in the extracellular environment. Docking experiments indicate the importance of an N-glycosylation site close to the active site for substrate specificity. Optimal pH and temperature for levan hydrolysis are 5.0 and 43.7°C, respectively. Docking experiments suggested multiple substrate binding sites and levan-mediated enzyme dimerization, explaining the observed positive cooperativity (Hill kinetics). Alignments show a one amino acid shift in the position of a conserved DXX(R/K) couple, typical for sucrose binding in cell wall invertases. A possible involvement of plant fructanases in levan trimming is discussed, in line with the emerging “fructan detour” concepts, suggesting that levan oligosaccharides act as signalling entities during plant microbial interactions.

Sweet Immunity Aspects during Levan Oligosaccharide-Mediated Priming in Rocket against Botrytis cinerea

“…Levan of microbial origin is the only potential substrate for these enzymes. Recently, a chicory fructanase with the ability to degrade levan was studied, and the enzyme was shown to localize to the apoplast [88]. In conclusion, the action of apoplastic 6-FEHs, in concert with bacterial endo-levanases, may increase LOS production rates.…”

“…Fructan degradation requires action of microbial endo-fructanases, plant apoplastic FEHs, or both. 6-FEHs with levan-hydrolyzing activity have been identified in several non-fructan plants [85][86][87], and an apoplastic localization has been confirmed for a chicory 6-FEH recently [88], further confirming a putative function in degrading microbial levan into LOS, acting as prebiotics and/or signaling compounds in the plant rhizosphere [84,88]. Possibly, similar mechanisms may be at play in the plant phyllosphere as well.…”

Fructan exohydrolases (FEHs) are upregulated by salicylic acid together with defense‐related genes in non‐fructan accumulating plants

“…Under stress, FEH activity was also detected in the apoplast together with fructans in response to cold in oat ( Avena sativa ; Livingston & Henson, 1998) and wheat ( Triticum aestivum ; Van den Ende et al, 2005). Recently, an apoplastic 6‐FEH was characterized in the inulin‐accumulating species Cichorium intybus (Ci6‐FEH II; Versluys, Porras‐Domínguez, et al, 2022). Unlike 1‐FEHs found in this species, the Ci6‐FEH II has little to no affinity for endogenous inulins and acts instead toward microbial levans.…”

Different evolutionary pathways to generate plant fructan exohydrolases

“…FEHs belong to the GH32 family. When they cut off terminal fructose moieties from inulin, levan, or graminan, they are termed fructan 1-exohydrolase (1-FEH), fructan 6-exohydrolase (6-FEH), and 6&1-FEH, respectively ( Versluys et al , 2022 ). Recently, a 6G&1-FEH, able to degrade neo-inulin-type fructans, was discovered in asparagus ( Ueno et al , 2018 ).…”

“…Recently, a 6G&1-FEH, able to degrade neo-inulin-type fructans, was discovered in asparagus ( Ueno et al , 2018 ). 1-FEHs have so far only been described in fructan-accumulating plants, while 6-FEHs have been identified in several non-fructan plants where they may be involved in the degradation of fructans from levan-producing plant pathogens or rather act as fructan trimmers on levans originating from beneficial bacteria ( Tian et al , 2021 ; Versluys et al , 2022 ). In the late 1990s, after the first plant FTs were cloned, the hunt was on to clone the first plant FEHs.…”