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Fructans (polyfructosylsucrose) are water-soluble and nonstructural polysaccharides consisting of linear or branched fructose chains attached to a sucrose moiety and are found in approx. 15% of flowering plant species (Hendry & Wallace, 1993). They are important storage carbohydrates in plants such as Poaceae (e.g. wheat and barley), Asteraceae (e.g. chicory, Jerusalem artichoke and Cynara cardunculus) and Liliaceae (e.g. onion and asparagus) (Suzuki, 1993; Raccuia & Melilli, 2004), and are synthesized in vacuoles (Frehner et al., 1984; Darwen & John, 1989). Fructans are considered to contribute to drought and cold resistance (Hendry, 1993; Pilon-Smits et al., 1995) and snow mold resistance (Yoshida et al., 1998).
We previously reported that asparagus (Asparagus officinalis) and onion (Allium cepa) plants contain two types of fructans distinguished from each other by their structures (Shiomi, 1989). One is an inulin-type fructan (1F(1-β-d-fructofuranosyl)m sucrose) which is a β-2,1-linked fructose-oligomer or -polymer terminated by glucose. The other is a fructan (1F(1-β-d-fructofuranosyl)m-6G(1-β-d-fructofuranosyl)n sucrose) derived from neokestose (6G-β-d-fructofuranosylsucrose, 6G-kestotriose) which has β-2,1-linked fructosyl residue(s) on the carbon-6 of the terminal glucosyl residue of inulin-type fructan. The latter, called fructan of the inulin neoseries, is usually found in liliaceous plants. These saccharides are synthesized by activities of sucrose:sucrose 1-fructosyltransferase (1-SST, EC 18.104.22.168), fructan:fructan 1-fructosyltransferase (1-FFT, EC 22.214.171.124) and fructan:fructan 6G-fructosyltransferase (6G-FFT or 6G-fructosyltransferase, 6G-FT) (Shiomi, 1989). 1-SST synthesizes 1-kestose (1-β-d-fructofuranosylsucrose, 1-kestotriose), an inulin-type trisaccharide, from two molecules of sucrose by fructosyltransfer (Edelman & Jefford, 1968; Shiomi & Izawa, 1980; Koops & Jonker, 1996; Lüscher et al., 1996). 1-FFT elongates the fructose chain of inulin-type fructans by fructosyltransfer from 1-kestose to another 1-kestose or fructan (Edelman & Jefford, 1968; Shiomi, 1982a; Lüscher et al., 1993; Koops & Jonker, 1994; van den Ende et al., 1996). 6G-FFT catalyses the transfer of a fructosyl residue from 1-kestose to carbon-6 of the terminal glucosyl moiety of sucrose or inulin-type fructan, producing neokestose or inulin neoseries fructan with a higher degree of polymerization (DP), respectively (Shiomi, 1981). It is a key enzyme in the biosynthesis of fructan of the inulin neoseries in asparagus and onion plants (Shiomi, 1989; Wiemken et al., 1995; Vijn & Smeekens, 1999; Ritsema et al., 2003; Fujishima et al., 2005).
Recently, many studies with molecular cloning have been performed to investigate the evolution and the function of fructosyltransferases in fructan biosynthesis, and to improve biological potentials of plants by enzymes such as 1-SST (de Halleux & van Cutsem, 1997; Hellwege et al., 1997), 1-FFT (Hellwege et al., 1998; van der Meer et al., 1998), 6G-FFT (Vijn et al., 1997) and sucrose:fructan 6-fructosyltransferase (6-SFT; Sprenger et al., 1995; Kawakami & Yoshida, 2002). Deduced primary sequences of these enzymes and invertases are classified in glycoside hydrolase family 32 (Henrissat, 1991, http://afmb.cnrs-mrs.fr/CAZY/acc.html). A cDNA encoding 6G-FFT from onion has been reported as a 6G-FFT gene (Vijn et al., 1997). The onion recombinant enzyme had 1-FFT activity with 6G-FFT activity, which agreed with our results with native 6G-FFT purified from onion bulbs (Fujishima et al., 2005). However, 6G-FFT purified from asparagus roots (Shiomi, 1981) showed high 6G-FFT activity, while no activity of invertase, 1-SST and 1-FFT were detected under defined reaction conditions (reaction with 0.2 m 1-kestose at pH 5.5 and 30°C for 1 h). Thus, we were interested in cloning a cDNA encoding 6G-FFT from asparagus, which would show different enzymatic characteristics from onion enzymes. The aim of the present study is to obtain information on 6G-FFT from asparagus based on nucleotide sequence and to elucidate enzymatic characteristics of the protein involved in fructan metabolism.
Here, we describe isolation of a cDNA clone encoding 6G-FFT from asparagus leaves and expression in the methylotrophic yeast Pichia pastoris. Characteristics of an enzyme preparation obtained by using a transformant harboring 6G-FFT cDNA from asparagus support the notion that the enzyme system in asparagus differs from that in onion.
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The present study shows that a cDNA clone named aoft1 encoding 6G-FFT has been isolated from a cDNA library of asparagus leaves and that a recombinant protein has been successively expressed in P. pastoris. The aoft1 encoded a protein named AoFT1 consisted of 610-amino acids, and the molecular mass and pI of the protein were calculated to be 68311 Da and 5.4, respectively. The deduced amino acid sequences of aoft1 showed the greatest identity (68%) and similarity (79%) to those of onion 6G-FFT. The N-terminal amino acids of mature 6G-FFTs from both asparagus and onion are not clear. Vijn et al. (1997) compared their results with barley 6-SFT (Sprenger et al., 1995) and carrot vacuolar invertase (Unger et al., 1994). Homology of the primary sequence of onion 6G-FFT with those sequences started at the 58th codon, and onion 6G-FFT was suggested to be made from a larger precursor protein containing a signal (for vacuolar-targeting) peptide located at N-terminus. The putative signal peptides in the three sequences were also revealed to be less conserved with one another. In the first 52-amino acid sequences of N-terminus of AoFT1, 18 amino acid residues were identical with those of onion 6G-FFT and other 12 amino acid residues were conservatively substituted, indicating that AoFT1 also has a signal peptide consisting of around 52 amino acids. This is expected for AoFT1, because the synthesis of fructan in plants is known to be located in vacuole (Frehner et al., 1984; Darwen & John, 1989).
The glycoside hydrolase family 32 (Henrissat, 1991, http://afmb.cnrs-mrs.fr/CAZY/acc.html) contains enzymes such as invertase, FEH, 1-SST, 1-FFT, 6G-FFT and 6-SFT. These enzymes are considered to have evolved from invertases. Moreover, primary structures of enzymes catalysing the different transfructosylation in liliaceous plant are more closely related to one another than those of enzymes catalysing the same reaction in asteraceous and gramineous plants (Fig. 2).
Recombinant AoFT1 protein obtained from P. pastoris showed 6G-FFT activity. Although four recombinant proteins, named AoFT1-NM1, AoFT1-NS50, AoFT1-NV53, and AoFT1-NA58, respectively, showed nearly the same patterns of products (data not shown), deletion of N-terminal sequences of the protein had an effect on the amount of enzymatic activity contained in the culture broth from Pichia transformants (Fig. 4). The effect may suggest that a deletion of N-terminal amino acid sequences at an appropriate position in AoFT1 is important for secretion of a protein into the culture broth. A similar result, that addition of an original signal peptide decreased enzyme activity in the culture broth, was reported in expression of barley 6-SFT in Pichia (Hochstrasser et al. 1998). However, the possibility that the signal or prosequence interfered with the enzyme activity still remained, because the enzyme has not yet been purified. To clarify this, purification of enzymes with different N-terminal sequence is needed.
The recombinant enzyme had not only 6G-FFT activity but also 1-FFT activity. The ratio of the activity of 6G-FFT to 1-FFT in the recombinant 6G-FFT from asparagus was calculated to be 13. The recombinant onion 6G-FFT from stable transformed tobacco plants also catalysed the production of the same saccharides including nystose from 1-kestose (Vijn et al., 1997) and Ritsema et al. (2003) demonstrated that the recombinant 6G-FFT from onion was a bifunctional enzyme that showed both 6G-FFT and 1-FFT activity. In purified native 6G-FFT from onion, the ratio of the 6G-FFT activity to 1-FFT in the native enzyme was calculated to be 2.3 (Fujishima et al., 2005). The 6G-FFT from onion was distinguished from the asparagus enzyme by relatively high 1-FFT activity. With regard to 1-FFT activity of 6G-FFT from asparagus, the native 6G-FFT from asparagus roots (Shiomi, 1981) produced only a trace of nystose from 1-kestose in prolonged reaction. Although the reason for the difference in 1-FFT activity of 6G-FFT between the native and the recombinant 6G-FFT from asparagus is not clear, most of 1-FFT activity of the recombinant 6G-FFT from asparagus may result from the expression system in P. pastoris. Indeed, a barley 6-SFT expressed in P. pastoris was reported to show unexpected 1-SST activity, and it was described that the differences in enzymatic characteristics between the native and recombinant 6-SFT might result from to difference in folding or glycosylation between the Pichia and the plant system (Hochstrasser et al., 1998).
In asparagus, three enzymes, 1-SST, 1-FFT and 6G-FFT participate in syntheses of fructans (Shiomi, 1989). The recombinant 6G-FFT from asparagus successively produced 4c and neokestose from 1-kestose, and showed the reverse reaction from neokestose (Figs 5 and 6). The properties of the recombinant 6G-FFT were similar to the native enzyme (Shiomi, 1981, 1982b). The enzyme also synthesized corresponding fructans of inulin neoseries from β-2,1-linked fructooligosaccharides (DP 5-9) from Jerusalem artichoke (Fig. 8). Effects of pH and temperature on the activities of both the native and the recombinant 6G-FFTs from asparagus agreed well with each other. The AoFT1 might actually play an important role in fructan metabolism of asparagus, acting as 6G-FFT in the three-enzyme system. In onion plant, however, no 1-FFTs have been purified or 1-FFT cDNAs cloned, although recombinant 6G-FFT has 1-FFT activity (Vijn et al., 1997; Ritsema et al., 2003). These results suggest that the three-enzyme system of fructan synthesis in asparagus may differ from the two-enzyme system in onion.
It remains unknown which amino acid residues are responsible for the differences of 1-FFT activity between asparagus 6G-FFT and the onion enzyme, while the deduced primary sequence of AoFT1 shows 68% identity to that of onion 6G-FFT. The same problems arise in fructosyltranferases involved in fructan metabolism, with high identities of primary sequences, but with a range of different enzymatic characteristics. To resolve these questions, further investigations are needed on the structures of domains in the enzymes corresponding to the binding of donor and acceptor saccharides.