Influencing the binding configuration of sucrose in the active sites of chicory fructan 1-exohydrolase and sugar beet fructan 6-exohydrolase

The hydrolytic plant enzymes of family 32 of glycoside hydrolases (GH32), including acid cell wall type invertases (EC 3.2.1.26), fructan 1-exohydrolases (1-FEH; EC 3.2.1.153) and fructan 6-exohydrolases (6-FEH; EC 3.2.1.154), are very similar at the molecular and structural levels, but are clearly functionally different. The work presented here aims at understanding the evolution of enzyme specificity and functional diversity in this family by means of site-directed mutagenesis. It is demonstrated for the first time that invertase activity can be introduced in an S101L mutant of chicory (Cichorium intybus) 1-FEH IIa by influencing the orientation of Trp 82. At high sucrose and enzyme concentrations, a shift is proposed from a stable inhibitor configuration to an unstable substrate configuration. In the same way, invertase activity was introduced in Beta vulgaris 6-FEH by introducing an acidic amino acid in the vicinity of the acid-base catalyst (F233D mutant), creating a beta-fructofuranosidase type of enzyme with dual activity against sucrose and levan. As single amino acid substitutions can influence the donor substrate specificity of FEHs, it is predicted that plant invertases and FEHs may have diversified by introduction of a very limited number of mutations in the common ancestor.     

X-ray diffraction structure of a plant glycosyl hydrolase family 32 protein: fructan 1-exohydrolase IIa of Cichorium intybus

Fructan 1-exohydrolase, an enzyme involved in fructan degradation, belongs to the glycosyl hydrolase family 32. The structure of isoenzyme 1-FEH IIa from Cichorium intybus is described at a resolution of 2.35 A. The structure consists of an N-terminal fivefold beta-propeller domain connected to two C-terminal beta-sheets. The putative active site is located entirely in the beta-propeller domain and is formed by amino acids which are highly conserved within glycosyl hydrolase family 32. The fructan-binding site is thought to be in the cleft formed between the two domains. The 1-FEH IIa structure is compared with the structures of two homologous but functionally different enzymes: a levansucrase from Bacillus subtilis (glycosyl hydrolase family 68) and an invertase from Thermotoga maritima (glycosyl hydrolase family 32).     

Hydrolase and fructosyltransferase activities implicated in the accumulation of different chain size fructans in three Asteraceae species.

Fructans are widely distributed in Asteraceae from floras with seasonal growth and are thought to be involved in drought and freezing tolerance, in addition to storage function. Reserve organs of Vernonia herbacea and Viguiera discolor, from the cerrado, and of the perennial herb Smallanthus sonchifolius, endemic to Andean region, store over 80% inulin, with different DP (35, 150, and 15, respectively). The fructan pattern in Asteraceae species could be explained by characteristics of their respective 1-FFTs. Hydrolases and fructosyltransferases from S. sonchifolius, V. herbacea and V. discolor were analyzed in plants at the same environmental conditions. The higher 1-FEH activities found in the species with lower DP, S. sonchifolius and V. herbacea reinforce the hypothesis of the involvement of 1-FEH in fructan profile and suggest that the high DP fructan of V. discolor is a consequence of the low affinity of its 1-FEH to the native long chain inulin. Long term incubation with sucrose suggested that the affinity of 1-FFT of V. discolor for 1-kestose is low when compared to that of V. herbacea. Indeed 1-FFT from V. discolor was shown to be an hDP 1-FFT, preferring longer inulins as acceptors. Conversely, 1-FFT from V. herbacea seems to have a higher affinity for short fructo-oligosaccharides, including 1-kestose, as acceptor substrates. Differences in fructan enzymes of the three Asteraceae provide new information towards the understanding of fructan metabolism and control of carbon flow between low and high DP fructans.     

Unraveling the difference between invertases and fructan exohydrolases: a single amino acid (Asp-239) substitution transforms Arabidopsis cell wall invertase1 into a fructan 1-exohydrolase

Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/FEHs.     

Cloning and functional analysis of a high DP fructan:fructan 1-fructosyl transferase from Echinops ritro (Asteraceae): comparison of the native and recombinant enzymes

Inulin-type fructans are the simplest and most studied fructans and have become increasingly popular as prebiotic health-improving compounds. A natural variation in the degree of polymerization (DP) of inulins is observed within the family of the Asteraceae. Globe thistle (Echinops ritro), artichoke (Cynara scolymus), and Viguiera discolor biosynthesize fructans with a considerably higher DP than Cichorium intybus (chicory), Helianthus tuberosus (Jerusalem artichoke), and Dahlia variabilis. The higher DP in some species can be explained by the presence of special fructan:fructan 1-fructosyl transferases (high DP 1-FFTs), different from the classical low DP 1-FFTs. Here, the RT-PCR-based cloning of a high DP 1-FFT cDNA from Echinops ritro is described, starting from peptide sequence information derived from the purified native high DP 1-FFT enzyme. The cDNA was successfully expressed in Pichia pastoris. A comparison is made between the mass fingerprints of the native, heterodimeric enzyme and its recombinant, monomeric counterpart (mass fingerprints and kinetical analysis) showing that they have very similar properties. The recombinant enzyme is a functional 1-FFT lacking invertase and 1-SST activities, but shows a small intrinsic 1-FEH activity. The enzyme is capable of producing a high DP inulin pattern in vitro, similar to the one observed in vivo. Depending on conditions, the enzyme is able to produce fructo-oligosaccharides (FOS) as well. Therefore, the enzyme might be suitable for both FOS and high DP inulin production in bioreactors. Alternatively, introduction of the high DP 1-FFT gene in chicory, a crop widely used for inulin extraction, could lead to an increase in DP which is useful for a number of specific industrial applications. 1-FFT expression analysis correlates well with high DP fructan accumulation in vivo, suggesting that the enzyme is responsible for high DP fructan formation in planta.     

Cloning, characterization and functional analysis of a 1-FEH cDNA from Vernonia herbacea (Vell.) Rusby

Variations in the inulin contents have been detected in rhizophores of Vernonia herbacea during the phenological cycle. These variations indicate the occurrence of active inulin synthesis and depolymerization throughout the cycle and a role for this carbohydrate as a reserve compound. 1-Fructan exohydrolase (1-FEH) is the enzyme responsible for inulin depolymerization, and its activity has been detected in rhizophores of sprouting plants. Defoliation and low temperature are enhancer conditions of this 1-FEH activity. The aim of the present work was the cloning of this enzyme. Rhizophores were collected from plants induced to sprout, followed by storage at 5 degrees C. A full length 1-FEH cDNA sequence was obtained by PCR and inverse PCR techniques, and expressed in Pichia pastoris. Cold storage enhances FEH gene expression. Vh1-FEH was shown to be a functional 1-FEH, hydrolyzing predominantly beta-2,1 linkages, sharing high identity with chicory FEH sequences, and its activity was inhibited by 81% in the presence of 10 mM sucrose. In V. herbacea, low temperature and sucrose play a role in the control of fructan degradation. This is the first study concerning the cloning and functional analysis of a 1-FEH cDNA of a native species from the Brazilian Cerrado. Results will contribute to understanding the role of fructans in the establishment of a very successful fructan flora of the Brazilian Cerrado, subjected to water limitation and low temperature during winter.     

Molecular cloning and characterization of a high DP fructan: fructan 1-fructosyl transferase from Viguiera discolor (Asteraceae) and its heterologous expression in Pichia pastoris

Inulin-type fructans are stored in the tuberous roots of the Brazilian cerrado plant Viguiera discolor Baker (Asteraceae). In Cynara scolymus (artichoke) and Echinops ritro (globe thistle), the fructans have a considerably higher degree of polymerization (DP) than in Cichorium intybus (chicory) and Helianthus tuberosus (Jerusalem artichoke). It was shown before that the higher DP in some species can be attributed to the properties of their fructan: fructan 1-fructosyl transferases (1-FFTs; EC 2.4.1.100), enzymes responsible for chain elongation. Here, we describe the cloning of a high DP (hDP) 1-FFT cDNA from V. discolor and its heterologous expression in Pichia pastoris . Starting from 1-kestose and Neosugar P (a mixture of oligo-inulins from microbial origin) as substrates, the recombinant enzyme produces a typical hDP inulin profile in vitro, closely resembling the one observed in vivo. The enzyme shows no invertase activity and sucrose: sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) activity in vitro. Pattern evolution during incubation suggests that inulins with DP ≥ 6 are much better substrates than sucrose or lower DP oligo-fructans. Because hDP inulin-type fructans show superior properties for specific food and non-food applications, the hDP 1-FFT gene from V. discolor has potential for the production of hDP inulin in vitro or in transgenic crops.     

Comparison of fructan dynamics in two wheat cultivars with different capacities of accumulation and remobilization under drought stress

Remobilization of stored carbohydrates in the stem of wheat plants is an important contributor to grain filling under drought stress (DS) conditions. A massive screening on Iranian wheat cultivars was performed based on stem dry weight changes under well-watered and DS conditions. Two cultivars, Shole and Crossed Falat Hamun (CFH), with different fructan accumulation and remobilization behavior were selected for further studies. Water-soluble carbohydrates (WSCs) and fructan metabolizing enzymes were studied both in the stem penultimate and in sucrose (Suc) treated, excised leaves. Under drought, CFH produced higher grain yields than Shole (412 vs 220 g m(-2)). Also, grain yield loss under drought was more limited in CFH than in Shole (17 vs 54%). Under drought, CFH accumulated more graminan-type fructo-oligosaccharides than Shole. After anthesis, fructan 6-exohydrolase (6-FEH; EC 3.2.1.154) activities increased more prominently than fructan 1-exohydrolase (EC 3.2.1.153) activities during carbon remobilization. Interestingly, CFH showed higher 6-FEH activities in the penultimate than Shole. The field experiment results suggest that the combined higher remobilization efficiency and high 6-FEH activities in stems of wheat could contribute to grain yield under terminal drought. Similar to the penultimate, fructan metabolism differed strongly in Suc-treated detached leaves of selected cultivars. This suggests that variation in the stem fructan among wheat cultivars grown in the field could be traced by leaf blade induction experiments.     

Fructan metabolising enzymes in rhizophores of Vernonia herbacea upon excision of aerial organs.

The activities of fructan metabolising enzymes and fructan contents are reported for rhizophores of Vernonia herbacea (Vell.) Rusby induced to sprouting by shoot excision. The activities of fructan exohydrolase (1-FEH), sucrose: sucrose fructosyltransferase (1-SST), fructan: fructan fructosyltransferase (1-FFT) and invertase (INV) and the fructan contents were analysed every 3-4 days for 1 month by colorimetric and chromatographic methods. Sprouting of new shoots started on day 9. 1-FEH activity increased after day 13 and reached its maximum value 20 days after shoot excision. A gradual decrease in 1-SST activity was detected between days 3 and 9. 1-FFT activity exhibited fluctuations throughout the experimental period and a peak of activity for invertase was detected 9 days after shoot excision. Variation in fructan contents in vivo included a decrease until day 13 after which, levels remained practically unchanged. Fructan depolymerization and sprouting are concomitant processes in V. herbacea and can be induced by shoot excision at any phenological phase. 1-FEH and 1-FFT seemed to act in a concerted way to catalyse fructan depolymerization, while 1-SST was inhibited, possibly due to interruption of sucrose supply to rhizophores from the aerial organs.     

Cloning and functional analysis of chicory root fructan1‐exohydrolase I (1‐FEH I): a vacuolar enzyme derivedfrom a cell‐wall invertase ancestor? Mass fingerprint of the 1‐FEH I enzyme

This paper describes the cloning and functional analysis of chicory (Cichorium intybus L.) fructan 1-exohydrolase I cDNA (1-FEH I). To our knowledge it is the first plant FEH cloned. Full-length cDNA was obtained by a combination of RT-PCR, 5' and 3' RACE using primers based on N-terminal and conserved amino acid sequences. Electrophoretically purified 1-FEH I enzyme was further analyzed by in-gel trypsin digestion followed by matrix-assisted laser desorption ionization and electrospray time-of-flight tandem mass spectrometry. Functionality of the cDNA was demonstrated by heterologous expression in potato tubers. 1-FEH I takes a new, distinct position in the phylogenetic tree of plant glycosyl hydrolases being more homologous to cell-wall invertases (44-53%) than to vacuolar invertases (38-41%) and fructosyl transferases (33-38%). The 1-FEH I enzyme could not be purified from the apoplastic fluid at significantly higher levels than can be explained by cellular leakage. These and other data suggest a vacuolar localization for 1-FEH I. Also, the pI of the enzyme (6.5) is lower than expected from a typical cell-wall invertase. Unlike plant fructosyl transferases that are believed to have evolved from a vacuolar invertase, 1-FEH I might have evolved from a cell-wall invertase-like ancestor gene that later obtained a vacuolar targeting signal. 1-FEH I mRNA quantities increase in the roots throughout autumn, and especially when roots are stored at low temperature.     

Fructan 1-exohydrolases. beta-(2,1)-trimmers during graminan biosynthesis in stems of wheat? Purification,characterization, mass mapping,and cloning of two fructan 1-exohydrolase isoforms

Graminan-type fructans are temporarily stored in wheat (Triticum aestivum) stems. Two phases can be distinguished: a phase of fructan biosynthesis (green stems) followed by a breakdown phase (stems turning yellow). So far, no plant fructan exohydrolase enzymes have been cloned from a monocotyledonous species. Here, we report on the cloning, purification, and characterization of two fructan 1-exohydrolase cDNAs (1-FEH w1 and w2) from winter wheat stems. Similar to dicot plant 1-FEHs, they are derived from a special group within the cell wall-type invertases characterized by their low isoelectric points. The corresponding isoenzymes were purified to electrophoretic homogeneity, and their mass spectra were determined by quadrupole-time-of-flight mass spectrometry. Characterization of the purified enzymes revealed that inulin-type fructans [beta-(2,1)] are much better substrates than levan-type fructans [beta-(2,6)]. Although both enzymes are highly identical (98% identity), they showed different substrate specificity toward branched wheat stem fructans. Although 1-FEH activities were found to be considerably higher during the fructan breakdown phase, it was possible to purify substantial amounts of 1-FEH w2 from young, fructan biosynthesizing wheat stems, suggesting that this isoenzyme might play a role as a beta-(2,1)-trimmer throughout the period of active graminan biosynthesis. In this way, the species and developmental stage-specific complex fructan patterns found in monocots might be determined by the relative proportions and specificities of both fructan biosynthetic and breakdown enzymes.     

Insights into the fine architecture of the active site of chicory fructan 1-exohydrolase: 1-kestose as substrate vs sucrose as inhibitor

* 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 K(i) (inhibition constant) well below physiological sucrose concentrations and could be rapidly activated under carbon deprivation.     

Sink filling, inulin metabolizing enzymes and carbohydrate status in field grown chicory (Cichorium intybus L.)

Inulin is a fructose-based polymer that is isolated from chicory (Cichorium intybus L.) taproots. The degree of polymerization (DP) determines its application and hence the value of the crop. The DP is highly dependent on the field conditions and harvest time. Therefore, the present study was carried out with the objective to understand the regulation of inulin metabolism and the process that determines the chain length and inulin yield throughout the whole growing season. Metabolic aspects of inulin production and degradation in chicory were monitored in the field and under controlled conditions. The following characteristics were determined in taproots: concentrations of glucose, fructose and sucrose, the inulin mean polymer length (mDP), yield, gene expression and activity of enzymes involved in inulin metabolism. Inulin synthesis, catalyzed by sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) (1-SST) and fructan:fructan 1-fructosyltransferase (EC 2.4.1.100) (1-FFT), started at the onset of taproot development. Inulin yield as a function of time followed a sigmoid curve reaching a maximum in November. Inulin reached a maximum mDP of about 15 in September, than gradually decreased. Based on the changes observed in the pattern of inulin accumulation, we defined three different phases in the growing season and analyzed product formation, enzyme activity and gene expression in these defined periods. The results were validated by performing experiments under controlled conditions in climate rooms. Our results show that the decrease in 1-SST that starts in June is not regulated by day length and temperature. From mid-September onwards, the mean degree of polymerization (mDP) decreased gradually although inulin yield still increased. The decrease in mDP combined with increased yield results from fructan exohydrolase activity, induced by low temperature, and the back transfer activity of 1-FFT. Overall, this study provides background information on how to improve inulin yield and quality in chicory.     

The genome structure of the <Emphasis Type="Italic">1-FEH</Emphasis> genes in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.): new markers to track stem carbohydrates and grain filling QTLs in breeding

Terminal drought tolerance of wheat is a major target in many areas in the world and is a particular focus in Western Australia. It is widely considered to relate to water soluble carbohydrate (WSC) levels such as fructan in the stem, as the head is maturing. Fructan exohydrolases are key enzymes during both fructan biosynthesis and mobilization. The wheat genome sequences of three fructan 1-exohydrolase (1-FEH) genes with seven exons and six introns were isolated by using the available 1-FEH w2 cDNA sequence. The major size differences among the three genes were located in intron 1 and intron 4. The three 1-FEH genes were mapped to Chinese Spring chromosome 6A, 6B and 6D based on polymerase chain reaction (PCR) polymorphisms and Southern hybridization. 1-FEH-6A, -6B and -6D corresponded to published cDNA sequences 1-FEH w1, w3 and w2, respectively. The overall correlation of the mRNA accumulation profile for the 1-FEH genes in stem and sheath leaf tissue in relation to the profile of soluble carbohydrate accumulation was consistent with their postulated role in stem soluble carbohydrate accumulation. The accumulation of the 1-FEH-6B (1-FEH w3) mRNA was 300 fold greater than that of 1-FEH-6A and -6D. The mRNA accumulation continued after the stem water soluble carbohydrate concentrations reached a peak, consistent with a role of 1-FEH-6B in the breakdown of soluble carbohydrate. The relationship between the 1-FEH genes and soluble carbohydrate accumulation is discussed and the 1-FEH-6B gene in particular is suggested to provide a new class of molecular marker for this trait. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.