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, 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.     

Factors affecting fructosyltransferases and fructan exohydrolase activities in <Emphasis Type="Italic">Agave tequilana</Emphasis> Weber var. azul

There is great interest in the fructosyltransferases (FTFs) involved in fructan metabolism and agents affecting their activity. Agaves accumulate fructans, fructose polymers linked by glycosidic β(2–1) and β(2–6) bonds in linear or branched configurations. In plants, fructans provide protection under stress conditions. The sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT), fructan:fructan 6G-fructosyltransferase (6G-FFT), and fructan exohydrolase (FEH) activities were analyzed in micropropagated Agave tequilana plants in the absence and presence of HgCl₂, AgNO₃, MgCl_2, sodium deoxycholate (DNa), and sodium dodecyl sulfate (SDS). Kestose, nystose and neokestose were synthesized by the respective FTFs. HgCl₂ and AgNO₃ inhibited all FTFs, mainly up to 90 % in 1-SST and 1-FFT. DNa increased 1-SST (32 %) and 1-FFT (45 %) activities, and SDS increased 6G-FFT activity by 96 %. Finally, AgNO₃ inhibited FEH activity by 78 %. Our results might be relevant on the regulation of FTFs in agave and other crops, for instance by the increment the fructans synthesis in stressed plants.     

Cloning of the fructan biosynthesis pathway of Jerusalem artichoke

To study the regulation of fructan synthesis in plants, we isolated two full-size cDNA clones encoding the two enzymes responsible for fructan biosynthesis in Jerusalem artichoke ( Helianthus tuberosus ): 1-sucrose:sucrose fructosyl transferase (1-SST) and 1-fructan:fructan fructosyl transferase (1-FFT). Both enzymes have recently been purified to homogeneity from Jerusalem artichoke tubers (Koops and Jonker (1994) J. Exp. Bot. 45, 1623–1631; Koops and Jonker (1996) Plant Physiol. 110, 1167–1175) and their amino acid sequences have been partially determined. Using RT–PCR and primers based on these sequences, specific fragments of the genes were amplified from tubers of Jerusalem artichoke. These fragments were used as probes to isolate the cDNAs encoding 1-SST and 1-FFT from a tuber-specific λZAP library. The deduced amino acid sequences of both cDNAs perfectly matched the sequences of the corresponding purified proteins. At the amino acid level, the cDNA sequences showed 61% homology to each other and 59% homology to tomato vacuolar invertase. Based on characteristics of the deduced amino acid sequence, the first 150 bp of both genes encode a putative vacuolar targeting signal. Southern blot hybridization revealed that both 1-SST and 1-FFT are likely to be encoded by single-copy genes. Expression studies based on RNA blot analysis showed organ-specific and developmental expression of both genes in growing tubers. Lower expression was detected in flowers and in stem. In other organs, including leaf, roots and dormant tubers, no expression could be detected. In tubers, the spatial and developmental expression correlates with the accumulation of fructans. Using the 1-sst and 1-fft cDNAs, chimeric genes were constructed driven by the CaMV 35S promoter. Analysis of transgenic petunia plants carrying these constructs showed that both cDNAs encode functional fructosyltransferase enzymes. Plants transformed with the 35S- 1-sst construct accumulated the oligofructans 1-kestose (GF₂), 1,1-nystose (GF₃) and 1,1,1-fructosylnystose (GF₄). Plants transformed with the 35S- 1-fft construct did not accumulate fructans, probably because of the absence of suitable substrates for 1-FFT, i.e. fructans with a degree of polymerization ≥ 3 (GF₂, GF₃, etc.). Nevertheless, protein extracts from these transgenic plants were able to convert GF₃, when added as a substrate, into fructans with a higher degree of polymerization. Progeny of crosses between a 35S- 1-sst -containing plant and a 35S- 1-fft- containing plant, showed accumulation of high-molecular-weight fructans in old, senescent leaves. Based on the comparison of the predicted amino acid sequences of 1-sst and 1-fft with those of other plant fructosyl transferase genes, we postulate that both plant fructan genes have evolved from plant invertase genes.     

Unexpected presence of fructan 6-exohydrolases (6-FEHs) in non-fructan plants: characterization, cloning, mass mapping and functional analysis of a novel "cell-wall invertase-like" specific 6-FEH from sugar beet (Beta vulgaris L.)

About 15% of flowering plant species synthesize fructans. Fructans serve mainly as reserve carbohydrates and are subject to breakdown by plant fructan exohydrolases (FEHs), among which 1-FEHs (inulinases) and 6-FEHs (levanases) can be differentiated. This paper describes the unexpected finding that 6-FEHs also occur in plants that do not synthesize fructans. The purification, characterization, cloning and functional analysis of sugar beet (Beta vulgaris L.) 6-FEH are described. Enzyme activity measurements during sugar beet development suggest a constitutive expression of the gene in sugar beet roots. Classical enzyme purification followed by in-gel trypsin digestion and mass spectrometry (quadruple-time-of-flight mass spectrometry (Q-TOF) MS) led to peptide sequence information used in subsequent RT-PCR based cloning. Levan-type fructans (beta-2,6) are the best substrates for the enzyme, while inulin-type fructans (beta-2,1) and sucrose are poorly or not degraded. Sugar beet 6-FEH is more related to cell wall invertases than to vacuolar invertases and has a low iso-electric point (pI), clearly different from typical high pI cell wall invertases. Poor sequence homology to bacterial or fungal FEHs makes an endophytic origin highly unlikely. The functionality of the 6-FEH cDNA was further demonstrated by heterologous expression in Pichia pastoris. As fructans are absent in sugar beet, the role of 6-FEH in planta is not obvious. Like chitinases and beta-glucanases hydrolysing cell-surface components of fungal plant pathogens, a straightforward working hypothesis for further research might be that plant 6-FEHs participate in hydrolysis (or prevent the formation) of levan-containing slime surrounding endophytic or phytopathogenic bacteria.     

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.     

Low temperature and defoliation affect fructan-metabolizing enzymes in different regions of the rhizophores of Vernonia herbacea

In addition to the storage function, fructans in Asteraceae from floras with seasonal growth have been associated with drought and freezing tolerance. Vernonia herbacea, native of the Brazilian Cerrado, bears underground reserve organs, rhizophores, accumulating inulin-type fructans. The rhizophore is a cauline branched system with positive geotropic growth, with the apex (distal region) presenting younger tissues; sprouting of new shoots occurs by development of buds located on the opposite end (proximal region). Plants induced to sprouting by excision of the aerial organs present increased 1-fructan exohydrolase (1-FEH) activity in the proximal region, while plants at the vegetative stage present high 1-sucrose:sucrose fructosyltransferase (1-SST) in the distal region. The aim of the present study was to analyze how low temperature (5 °C) could affect fructan-metabolizing enzymes and fructan composition in the different regions of the rhizophores of intact and excised plants. 1-SST and 1-fructan:fructan fructosyltransferase (1-FFT) were higher in the distal region decreasing towards the proximal region in intact plants at the vegetative phase, and were drastically diminished when cold and/or excision were imposed. In contrast, 1-FEH increased in the proximal region of treated plants, mainly in excised plants subjected to cold. The ratio fructo-oligo to fructo-polysaccharides was significantly higher in plants exposed to low temperature (1.17 in intact plants and 1.64 in excised plants) than in plants exposed to natural temperature conditions (0.84 in intact vegetative plants and 0.58 in excised plants), suggesting that oligosaccharides are involved in the tolerance of plants to low temperature via 1-FEH, in addition to 1-FFT. Principal component analysis indicated different response mechanisms in fructan metabolism under defoliation and low temperature, which could be interpreted as part of the strategies to undergo unfavorable environmental conditions prevailing in the Cerrado during winter.     

Crystal structures of Arabidopsis thaliana cell-wall invertase mutants in complex with sucrose

In plants, cell-wall invertases fulfil important roles in carbohydrate partitioning, growth, development and crop yield. In this study, we report on different X-ray crystal structures of Arabidopsis thaliana cell-wall invertase 1 (AtcwINV1) mutants with sucrose. These structures reveal a detailed view of sucrose binding in the active site of the wild-type AtcwINV1. Compared to related enzyme-sucrose complexes, important differences in the orientation of the glucose subunit could be observed. The structure of the E203Q AtcwINV1 mutant showed a complete new binding modus, whereas the D23A, E203A and D239A structures most likely represent the productive binding modus. Together with a hydrophobic zone formed by the conserved W20, W47 and W82, the residues N22, D23, R148, E203, D149 and D239 are necessary to create the ideal sucrose-binding pocket. D239 can interact directly with the glucose moiety of sucrose, whereas K242 has an indirect role in substrate stabilization. Most probably, K242 keeps D239 in a favourable position upon substrate binding. Unravelling the exact position of sucrose in plant cell-wall invertases is a necessary step towards the rational design of superior invertases to further increase crop yield and biomass production.     

An alternate sucrose binding mode in the E203Q Arabidopsis invertase mutant: an X-ray crystallography and docking study

In the present study, we report on the X-ray crystallographic structure of a GH32 invertase mutant, (i.e., the Arabidopsis thaliana cell-wall invertase 1-E203Q, AtcwINV1-mutant) in complex with sucrose. This structure was solved to reveal the features of sugar binding in the catalytic pocket. However, as demonstrated by the X-ray structure the sugar binding and the catalytic pocket arrangement is significantly altered as compared with what was expected based on previous X-ray structures on GH-J clan enzymes. We performed a series of docking and molecular dynamics simulations on various derivatives of AtcwINV1 to reveal the reasons behind this modified sugar binding. Our results demonstrate that the E203Q mutation introduced into the catalytic pocket triggers conformational changes that alter the wild type substrate binding. In addition, this study also reveals the putative productive sucrose binding modus in the wild type enzyme.     

Fructosyl transferase and hydrolase activities in rhizophores and tuberous roots upon growth of Polymnia sonchifolia (Asteraceae)

The underground reserve organs of yacon (Polymnia sonchifolia Poep. Endl.), similarly to other economically important Asteraceae, accumulate more than 60%, on a DW basis, of inulin type β(2‐1) fructans, mainly oligomers (GF2–GF16). Although sucrose:sucrose 1‐fructosyl transferase (1‐SST), fructan:fructan 1‐fructosyl transferase (1‐FFT) and fructan 1‐exohydrolase (1‐FEH) were properly described and characterized from a number of plant species, detailed information about their activities in different organs during development are rather scarce in the literature. In the present work 1‐SST, 1‐FFT and 1‐FEH activities were measured monthly in rhizophores and tuberous roots of yacon plants during their complete growth cycle under field conditions. Results showed that 1‐SST activity in rhizophores was always higher than 1‐FFT activity and increased up to 8 months of cultivation, decreasing to initial values at the end of the growth period. In the tuberous roots 1‐SST activity was also higher than 1‐FFT but varied differently. The higher values were found at the beginning of tuberization (3‐month‐old plants) and at the flowering phase (7‐month‐old plants). Results also showed that synthesizing activities in yacon plants were always higher in rhizophores than in the tuberous roots, while hydrolysing activity predominated in the latter, mainly when 1‐kestose and nystose were used as substrates. 1‐FEH from yacon plants showed low efficiency when commercial inulin from Helianthus tuberosus was utilized as substrate. The analysis of the enzymatic activities performed during growth of yacon clearly indicated the most appropriate source organ and phase of development to obtain the highest enzymatic activities for purification purposes and for the production of fructo‐oligosaccharides (FOS). Furthermore, the results suggested that the relative levels of activities of 1‐SST, 1‐FFT and 1‐FEH could be involved in the chain length distribution of the fructan molecules found in rhizophores and in tuberous roots of this species.     

Effect of defoliation on fructan pattern and fructan metabolizing enzymes in young chicory plants (Cichorium intybus)

Witloof chicory ( Cichorium intybus L. var. foliosum cv. Flash) was sown in acid-washed vermiculite in a controlled growth chamber. After 1 month of growth, one half of the chicory plants were defoliated whereas the intact chicory plants remained as a control. Twenty-four hours after defoliation, a very sharp decrease in hexose, sucrose, and total fructan concentration was observed in the roots. This coincided with a strong decrease in sucrose:sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99) activity and a strong increase in fructan 1-exohydrolase (1-FEH; EC 3.2.1.80) activity. After day 5, 1-SST activity increased and 1-FEH activity decreased. However, from day 5 to 15, both the activities of 1-SST and acid invertase (EC 3.2.1.26) remained significantly lower than in the control plants. From 10 days after defoliation, fructan synthesis resumed and hexose and sucrose concentrations increased. Up to now, 1-FEH activity was believed to occur only in mature tissues (end of the growing season, storage, forcing, or sprouting). Therefore, the rather unexpected finding that 1-FEH can also be induced in very young chicory roots after defoliation suggests that 1-FEH can be considered a 'survival' enzyme that can be induced at any physiological stage when energy demands increase.