Fructan Metabolism

Although fructose nucleotides (UDP-fructose and ADP-fructose) have been isolated from fructan synthesizing tissues (36), no evidence for their physiological role as fructose donors in fructan synthesis had been obtained. Edelman and Jefford (37) described the enzyme systems of sucrose sucrose fructosyl transferase (1-SST) and fructan fructan fructosyl transferase (1-FFT) for the synthesis of fructan. On storing chicory roots for forcing or sprouting of jerusalem artichoke tubers or during flowering in chicory, fructan exohydrolase (l-FEH) becomes active. Another important feature of fructan storing tissues is that glucose, the product of 1-SST, does not accumulate. Obviously glucose to sucrose conversion either by sucrose synthase (SS) or sucrose phosphate synthase (SPS) could be important. Possibly some of the sucrose is also hydrolyzed by invertase in these tissues.

3.1. Localization of enzymes of fructan metabolism

Inulin was suggested to be located in the vacuoles on the grounds of its high concentrations (up to 80 % of dry weight in jerusalem artichoke tubers), high water solubility and large volume of the cell occupied by the vacuole. It accumulates in the large central vacuole of the storage parenchyma (14, 37). To account for the biosynthesis of vacuolar fructan from cytosolic sucrose, Edelman and Jefford (37) proposed that SST was cytosolic and FFT was located in the tonoplast so that fructosyl residues were transferred from the cytosolic trisaccharide across the tonoplast to the elongating chains of fructan within the vacuole. Similarly they suggested that FEH could most effectively mobilize fructan if the enzyme was located at the tonoplast transferring fructosyl residues from vacuole to cytosol. This scheme though elegantly accounts for matin features of fructan synthesis but failed to find support in the work of Frehner et al (38) who concluded that both SST and FFT were located in the vacuole.

Frehner et al (38) analyzed the contents of the vacuoles isolated from the tuber protoplasts of jerusalem artichoke. They observed that during the course of preparation of vacuoles, enzymes of fructan metabolism co-

sedimented with the vacuole markers cc-mannosidase and p-N-acetyl glucosaminidase. Their results showed that isokestose and higher fructans and all anabolic and catabolic enzymes in inulin metabolism are located exclusively in the vacuoles. These results are in good agreement with the findings of the compartmentation of fructan metabolism in cereal leaves ( 39-40). FFT is the sole enzyme of inulin metabolism which is active during the whole life cycle of jerusalem artichoke tuber. The activity of SST is present only in developing tubers and that of FEH only in resting or sprouting tubers. In contrast fructose, glucose and sucrose appear to be located also outside the vacuole (38).

Edelman and Jefford (37) had shown that while SST has a high Km for sucrose, FFT catalyzed polymerization is effectively inhibited by sucrose. Because of the contrasting sucrose requirement of SST (high sucrose level) and FFT (low sucrose level), SST and FFT were allocated to cytosol and vacuole, respectively (37).

Darwen and John (41) observed that during vacuole purification from jerusalem artichoke tubers, the ratio of activities of SST and FFT to fructan and to a-mannosidase (vacuole marker) activity remained relatively constant. However, the ratio of their activities to cytosolic markers alcohol dehydrogenase and glucose-6-phsosphate dehydrogenase increased up to 5 folds. Thus they concluded that in Jerusalem artichoke tubers both SST and FFT are vacuolar enzymes (41). In order to determine whether the SST and FFT are located at the tonoplast or within the vacuole sap, the isolated vacuoles were lysed and separated into soluble and membrane fractions. Both SST and FFT activities were almost entirely associated with the vacuole sap. Similarly during the phase of fructan utilisation in the tubers, FFT and FEH activities got co-purified during vacuole isolation and both the activities resided in the vacuoles. While FFT activity was found in vacuole sap, the FEH activity was largely associated with the tonoplast. During depolymerization, fructan is hydrolysed by vacuole localized fructan exo hydrolase to fructose which can be exported to cytoplasm (41). However, in a recent paper, the exclusive vacuolar localization of fructan metabolism has been criticized and the presence of both the fructan and FEH are reported in the apoplastic fluid (42).

3.2. Enzymology of fructan metabolism 3.2.1. 1-SST

Sucrose sucrose fructosyl transferase (1-SST) is a key enzyme in the biosynthesis of inulin and it catalyses the synthesis of 1-kestose (GF2) from sucrose by trans fructosylation reaction. Activity of SST in roots of young plants of chicory synthesizing large amounts of fructans was high than in mature roots suggesting a role for 1-SST in determining sink strength (43). An enzyme purified from chicory root could accomplish the synthesis of GF2, while at low concentration it hydrolyzed sucrose therefore this enzyme was designated as invertase (44). 1-SST has been purified to homogeneity from tubers of H. tuberosus (45). In this study, dormant tubers, which donot actively accumulate fructans but do contain invertase were used as a source of protein extract. Furthermore, Praznik et al used 460 mM concentration of sucrose in the assay system (45). Under such conditions even a yeast invertase could accumulate GF2.

According to Cairns (46), the SST/FFT model for synthesis of fructan is not sufficiently proved because the enzymological evidences obtained were either with crude extracts or only partially purified preparations. Moreover in many cases the extracts were contaminated with invertase which because of its artificial fructosyl transferase activity could be equally responsible for fructosyl transferase activity.

1-SST was purified 665 folds from tubers of H. tuberosus (47). SDS dissociated 1-SST into 27- and 55-KD polypeptides. The 6-SFT (sucrose: fructan fructosyl transferase) purified from barley under denaturing conditions yielded 20- and 50-KD fragments (48). Evidences showed that the 27- and 55-KD fragments were derived from a single protein (47). Some of the fructose released by SST may also be due to the SST-mediated hydrolysis of oligofructans and that synthetic and hydrolytic activities may reside on the same protein (47).

1-SST and 1-FFT, the two enzymes needed to synthesize fructan, differ in their chromatographic and electrophoretic behaviours and also enzymic properties. For example 1-FFT is not able to catalyze the initial step of fructan synthesis whereas 1-SST is not able to catalyze the synthesis of fructan polymers with a DP higher than 5. However, 1-SST is able to transfer fructosyl units as effectively as 1-FFT between GF2, GF3 or GF4 molecules. Although 1-SST and 1-FFT have some overlapping activity (both enzymes can catalyze the formation of GF3 and GF4); GF3 and GF4 synthesis is more efficiently catalyzed by 1-FFT (49).

Despite the poorly balanced enzymatic properties of 1-SST and 1-FFT it is possible to synthesize fructans from sucrose after recombining purified 1-SST and 1-FFT. The synthesis of fructans with a DP up to 15 after 80 h of incubation with sucrose using these two enzymes was demonstrated (49). Fructosyl units are probably transferred between fructans with the same DP as can be concluded from the finding that GFn+1 is synthesized only after some GFn has accumulated (49).

On the basis of investigations of Koops and Jonker (49), it can be concluded that the basic concept of the early model of fructan synthesis proposed by Edelman and Jefford (37) in H. tuberosus, one enzyme for trisaccharide synthesis and one enzyme for the synthesis of higher fructans, is supported by experimental evidences and reservations on soundness of this model (46, 50) may not be valid at least in this crop.

1-SST has also been purified 63 folds from roots of chicory (51). The MW of 69 KD of chicory root SST is comparable MW of other purified 1-SST enzymes (45, 52, 53) and also with chicory root 1-FFT (54). Possibly 69 KD monomer is processed rapidly into 49 and 20 KD sub units in vivo since no 69 KD fragment as tagged on a western blot of SDS-boiled extracts. Although the pH and temperature optima and apparent Km values for chicory root 1-SST and other 1-SST preparations are similar to the characteristics of an invertase from yeast (50), an organism in which no fructans occur, but, this SST preparation was clearly different from yeast invertase in two aspects, (a) chicory root 1-SST only produces 1-kestose as a trisaccharide from sucrose whereas yeast invertase predominantly produces 6-kestose and lower amounts of neo kestose and 1-kestose (b) chicory root 1-SST mainly shows fructosyl transferase activity and no P-fructosidase activity even at low sucrose concentration

The fructose/1-kestose ratio after 1-SST reaction varies from 0.18 at 20 mM sucrose to 0.048 at 100 mM sucrose. Under these conditions yeast invertase shows almost exclusively p-fructosidase activity e.g. fructose/1-kestose ratio varied from about 100 at 20 mM sucrose to 40 at 100 mM sucrose (51). Therefore, chicory root 1-SST is not an invertase but a genuine 1-SST. However, some fructose is still produced, especially at low sucrose concentration and higher temperature. Low p-fructosidase activity is believed to be a real characteristic of the enzyme. This p-fructosidase activity at low sucrose concentration has also been reported to be a typical characteristic of other purified plant, bacterial or fungal fructosyl transferases (20, 55-61).

1-SST N-terminal has a high homology with 6-SFT from barley and some plant invertases (especially the vacuolar invertases). It has been demonstrated that bacterial levan sucrase can be converted into invertase by single point mutations (56). Therefore it might well be possible that plant fructosyl transferases have evolved from plant invertases by small mutational changes (51).

1-kestose can act as a donor for 1-SST. Therefore, during short incubations, the following reactions can occur in 1-SST/sucrose reaction mixture.

The possibility of following futile reaction can not be ruled out.

It is an intriguing question whether fructose produced as a function of time originates from reaction (b)or reaction (c) or both. Sucrose is a much better substrate than 1-kestose for 1-SST.

A discussion on the kinetics of 1-SST is not easy because the traditional Michaelis-Menten equation can, infect, only be used for one substrate reactions. 1-SST is a two substrate reaction where two identical substrates are involved in one reaction. If one assumes that 1-SST has two active sites: one for donor molecule and one for acceptor site (60), sigmoidal kinetics is expected unless the dissociation constants of both active sites are greatly different. In the case of 1-SST, both the donor and acceptor concentrations can not be varied independently and hence a calculation of Km becomes impossible (47).

The sucrose concentration in chicory juice is about 20 mM. However, the exact concentration at the place of fructan in situ is unknown and might be higher than 20 mM (51). At a sucrose concentration above 20 mM, the enzyme mainly acts as 1-SST and not as an invertase even in vitro. The fructan produced by 1-SST in vitro from sucrose are of inulin type. Probably the hydrolytic activities of 1-SST, reaction (b) and (c) are less important in vivo than in vitro since the in vivo sucrose concentration is likely to be held relatively constant by continuous supply from leaves while sucrose is rapidly depleted in vitro (51).

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