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J. Biol. Chem., Vol. 277, Issue 1, 194-200, January 4, 2002

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Chain Elongation of Raffinose in Pea Seeds

ISOLATION, CHARCTERIZATION, AND MOLECULAR CLONING OF A MULTIFUNCTIONAL ENZYME CATALYZING THE SYNTHESIS OF STACHYOSE AND VERBASCOSE^*,

Thomas Peterbauer, Jan Mucha^§, Lukas Mach^§, and Andreas Richter^¶

From the  Institute of Ecology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria and the ^§ Centre for Applied Genetics, University of Agricultural Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria

Received for publication, October 9, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Raffinose oligosaccharides are major soluble carbohydrates in seeds and other tissues of plants. Their biosynthesis proceeds by stepwise addition of galactose units to sucrose, which are provided by the unusual donor galactinol (O--D-galactopyranosyl-(11)-L-myo-inositol). Chain elongation may also proceed by transfer of galactose units between raffinose oligosaccharides. We here report on the purification, characterization, and heterologous expression of a multifunctional stachyose synthase (EC 2.4.1.67) from developing pea (Pisum sativum L.) seeds. The protein, a member of family 36 of glycoside hydrolases, catalyzes the synthesis of stachyose, the tetrasaccharide of the raffinose series, by galactosyl transfer from galactinol to raffinose. It also mediates the synthesis of the pentasaccharide verbascose by galactosyl transfer from galactinol to stachyose as well as by self-transfer of the terminal galactose residue from one stachyose molecule to another. These activities show optima at pH 7.0. The enzyme also catalyzes hydrolysis of the terminal galactose residue of its substrates, but is unable to initiate the synthesis of raffinose oligosaccharides by galactosyl transfer from galactinol to sucrose. A minimum reaction mechanism which accounts for the broad substrate specificity and the steady-state kinetic properties of the protein is presented.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sugars of the raffinose series consist of 1,6-linked chains of D-galactose attached to the 6-glucosyl position of sucrose. They are synthesized in leaves, roots, and tubers of a range of plant species. In seeds of higher plants, they are of almost ubiquitous occurrence (1). In some crop species, raffinose oligosaccharides comprise up to 16% of seed dry matter (1, 2). Aside from a role as storage and transport carbohydrates (3), other functions of these oligosaccharides remain elusive. In the specialized, desiccation-tolerant seeds of higher plants, raffinose and its higher homologues may play a role as protective agents during maturation drying (4). Furthermore, correlative and experimental data suggest they may act as cryoprotectants in frost-hardy plants (5, 6). While they have long been regarded as non-digestible factors promoting flatulence in human nutrition, more recent data suggest a beneficial effect of raffinose oligosaccharides on the gut microflora (7). Their physical characteristics make them also suitable for non-food applications, such as organ preservation (8).

The first step in the biosynthesis of raffinose oligosaccharides is the reversible transfer of the galactosyl residue of the unusual donor galactinol, an -galactoside of myo-inositol (O--D-galactopyranosyl-(11)-L-myo-inositol), to sucrose. The resulting trisaccharide raffinose serves as an acceptor for another galactosyl residue from galactinol, yielding the tetrasaccharide stachyose (for review, see Ref. 9). These reactions are catalyzed by raffinose synthase (EC. 2.4.1.82) and stachyose synthase (EC 2.4.1.67), respectively. myo-Inositol is recycled to galactinol by a specific galactosyltransferase utilizing UDP-galactose as donor (3). Very little information is available on the biochemistry of verbascose and ajugose, next higher homologues of the raffinose series. It is generally accepted that their biosynthesis involves galactinol as donor, but it is not clear whether there are specific transferases for each step (10). Verbascose synthase activity from seeds of Vicia faba co-purified with stachyose synthase activity during ammonium sulfate and calcium phosphate gel fractionation (11), while stachyose synthase purified to homogeneity from adzuki bean seeds was devoid of verbascose synthase activity (12). More recently, an alternative mode of chain elongation has been proposed, which does not involve galactinol (13). In leaves of Ajuga reptans, an enzyme activity has been identified which catalyzed transfer of the terminal galactose residue from one raffinose oligosaccharide molecule to another, resulting in the formation of molecules of the next higher and lower degree of polymerization. The enzyme was tentatively termed galactan:galactan galactosyltransferase (GGT).¹ Like verbascose synthase, GGT has not yet been purified or cloned. Activity was detected exclusively in vacuoles isolated from mesophyll protoplasts of A. reptans (14, 15). Consistent with its subcellular localization, GGT activity exhibited an acidic pH optimum (13), while the galactinol-dependent transferases display optima at neutral pH values.

We have previously detected a galactinol-dependent verbascose synthase activity as well as a GGT activity in crude enzyme extracts from pea seeds (16). In contrast to GGT activity from A. reptans, highest activity was observed at pH 7.0. These findings urged a more detailed study on the biosynthesis of verbascose in plant seeds. During attempts to purify the two transferases from pea seeds, we noticed that the activities co-purified with stachyose synthase. We here demonstrate that all activities are catalyzed by a single protein in pea seeds. A minimal reaction mechanism which accounts for the broad substrate specificity and steady-state kinetic properties of the enzyme is presented.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Seeds of pea (Pisum sativum L.) cv. Wunder von Kelvedon were obtained from a local supplier (Austrosaat, Vienna, Austria). Raffinose and myo-inositol were from Sigma. Stachyose was from Merck and was further purified by chromatography on charcoal-Celite (17). Verbascose was purchased from Megazyme. Galactinol (O--D-galactopyranosyl-(11)-L-myo-inositol), D-ononitol (1D-4-O-methyl-myo-inositol), and galactosyl ononitol (O--D-galactopyranosyl-(13)-4-O-methyl-D-myo-inositol) were available from previous studies (18, 19). D-Pinitol (1D-3-O-methyl-chiro-inositol) was obtained from Aldrich and sequoyitol (5-O-methyl-myo-inositol) was from Roth. Galactopinitol A (O--D-galactopyranosyl-(12)-4-O-methyl-D-chiro-inositol) was a gift of Dr. F. Keller (University of Zuerich, Switzerland).

Protein Purification-- All purification steps were carried out at 4 °C unless otherwise indicated. Maturing seeds were harvested 25 to 30 days after flowering, frozen in liquid nitrogen, and ground to a fine powder. The powder (280 g) was suspended in 1.5 ml of extraction buffer per gram of seeds and homogenized with a Polytron tissue homogenizer. The extraction buffer consisted of 50 mM HEPES-NaOH, pH 7.0, 20 mM MgCl₂, 2.5 mM EGTA, 0.5 mM dithiothreitol, and 1% polyvinyl polypyrrolidone. The homogenate was squeezed through a fine-mesh nylon cloth and centrifuged at 26,000 × g for 30 min. The supernatant was cleared by treatment with protamine sulfate (20) and fractionated with solid ammonium sulfate. Proteins precipitating between 35 and 55% saturation were collected by centrifugation at 26,000 × g for 30 min. The pellet was dissolved in 20 mM bis-Tris propane-HCl, pH 6.8, containing 0.5 mM dithiothreitol, and dialyzed overnight against this buffer. The dialyzed extract was applied to an anion-exchange column (2.5 × 18 cm) of Macro-Prep High Q support (Bio-Rad) thermostated at 12 °C. Bound protein was eluted with a linear gradient from 0 to 250 mM NaCl in 20 mM bis-Tris propane-HCl, pH 6.8, 0.5 mM dithiothreitol. Active fractions were pooled and concentrated by ultrafiltration (Centricon-plus 20, Millipore). Ammonium sulfate was added to a final concentration of 1.5 M and the sample was applied to a hydrophobic interaction chromatography column (2.5 × 5 cm) of Macro-Prep Methyl support (Bio-Rad). The column was thermostated at 12 °C. Bound protein was eluted by applying a linear gradient of 1.5 to 0 M ammonium sulfate in 50 mM HEPES-NaOH, pH 7.0, 0.5 mM dithiothreitol. Active fractions were concentrated by ultrafiltration and subjected to preparative PAGE using a Prep Cell (Model 491, Bio-Rad) as previously described (12). Active fractions were pooled, desalted, and concentrated by repeated ultrafiltration in 20 mM Na-phosphate, pH 6.8. The sample was applied to a column (0.8 × 2.4 cm) of ceramic hydroxyapatite (Macro-Prep CHT I, Bio-Rad) equilibrated with 10 mM Na-phosphate, pH 6.8. The column was maintained at 10 °C and bound protein was eluted with a linear gradient to 200 mM Na-phosphate, pH 6.8.

Enzyme and Protein Assay-- In standard reaction mixtures (20 µl), enzyme samples were incubated at 30 °C for 0.5 to 3 h in 20 mM Na-phosphate, pH 7.0, 0.5 mM dithiothreitol, containing either galactinol in combination with raffinose, galactinol in combination with stachyose, or stachyose alone (10 mM each). Enzyme dilutions and incubation times were chosen to allow transformation of not more than 10% of the substrates. Where specified, other galactosyl donors and acceptors were used. Reactions were stopped by incubation at 95 °C for 5 min. Samples were analyzed by HPAEC-PAD using a Dionex DX 500 chromatography system equipped with a Carbopac PA10 column (2 × 250 mm) and a PA10 guard column. The column was maintained at 25 °C and was eluted at a flow rate of 0.25 ml/min with the following NaOH gradient: 0-8 min, 50 mM; 8-24 min, 50-250 mM; 24-25 min, 250-50 mM; 25-30 min, 50 mM. For analysis of reaction products involving methylated inositols as galactosyl acceptors, reaction products were converted to trimethylsilyl derivatives and analyzed by capillary gas chromatography as previously described (18). Protein concentrations were determined by the Bradford dye binding procedure, using the Bio-Rad protein assay kit with bovine serum albumin as a standard.

SDS-PAGE, Western Blot Analysis, and Amino-terminal Protein Sequencing-- Samples were separated by SDS-PAGE on 7.5% acrylamide gels under reducing conditions. Gels were either stained with silver nitrate or blotted onto polyvinylidene difluoride membranes. The blots were probed with polyclonal rabbit antibodies to adzuki bean stachyose synthase as previously described (21) with modifications. The membranes were blocked overnight at 4 °C in TBS containing 5% non-fat milk, washed with TBS containing 0.5% Tween 20, and incubated for 1 h in blocking solution containing 0.5% Tween 20 and primary antibodies at a dilution of 1:5,000. Bound primary antibodies were visualized with alkaline phosphatase-conjugated goat anti-rabbit antibodies (Bio-Rad) and a chromogenic reaction using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates. For amino-terminal protein sequencing, the purified protein was electrophoresed as above, blotted, and stained with Coomassie Brilliant Blue. The sample was sequenced by automated Edman degradation at the Institute of Medicinal Biochemistry, University of Vienna, using an Applied Biosystems Model 476A sequencer.

Reverse Transcriptase-PCR and cDNA Cloning-- Total RNA was isolated from maturing seeds (about 25 days after flowering) with the RNeasy Plant Mini kit (Qiagen). Single-stranded cDNA was synthesized using Omniscript reverse transcriptase (Qiagen) in combination with an oligo(dT) primer. The cDNA was subjected to PCR with HotStarTaq DNA polymerase (Qiagen) in combination with degenerate primers. The sense primer (5'-GARMGIAARTTYAARGTIAARGG-3') was designed at 8 amino acids (ERKFKVKG) of the amino-terminal sequence of the purified protein. The antisense primer (5'-CCAICCISCICCYTGRCARTTRAA-3'), corresponding to the amino acid sequence FNCQG(A/G)GW, was based on a highly conserved box of the gene encoding for adzuki bean stachyose synthase and related sequences (21). The resulting PCR product was cloned into the pCR2.1-TOPO vector (Invitrogen) and sequenced using an ABI Prism BigDye Terminator Cycle Sequencing Mix and an ABI Prism 310 sequencer (Applied Biosystems). The full-length cDNA sequence was established by RNA ligase-mediated rapid amplification of cDNA ends using the Invitrogen GeneRacer^TM system. Briefly, total RNA was first treated with calf intestinal alkaline phosphatase to remove the 5'-phosphate groups of truncated mRNAs. Subsequently, 5'-phosphate groups of capped, full-length mRNAs were exposed by treatment with tobacco acid pyrophosphatase. A synthetic RNA oligonucleotide was ligated to the exposed phosphate groups, followed by first-strand synthesis with an oligo(dT) anchor primer. For amplification of the 5'-end, a primer at the RNA oligonucleotide was used with a gene-specific antisense primer (5'-TCAGGAACATCATGGAACAAAGGAA-3'). For amplification of the 3'-end, a gene-specific sense primer (5'-TGGGAAGAGTTGGGGATGATTTTTG-3') was used in combination with a primer homologous to the oligo(dT) primer, followed by PCR with a nested gene-specific primer (5'-GGGAAGTTTTTGGTTGCAAGGTGTG-3'). PCR products were cloned into the pCR2.1-TOPO vector and sequenced.

Heterologous Expression in Insect Cells-- The coding region of the putative stachyose synthase cDNA was amplified by reverse transcriptase-PCR using Pfu DNA polymerase (Promega) and the primer pair 5'-GCCGTCTAGACCATGGCTCCTCCC-3' and 5'-GCCAGATCTATGCAAGTAACACAAACACA-3'. Restriction sites for XbaI and BglII (underlined) were introduced with the primers for cloning of the PCR product into the XbaI-BglII site of the baculovirus expression vector pVL1393. The identity of the insert was verified by sequencing. The construct was co-transfected with BaculoGold viral DNA (PharMingen) into Spodoptera frugiperda Sf9 insect cells as previously described (22). After incubation for 5 days at 27 °C, the supernatant from the infected cells was collected and added to Sf21 cells for viral amplification. Infected cells were harvested, washed with phosphate-buffered saline containing a set of protease inhibitors (Complete protease inhibitor mixture, Roche Molecular Diagnostics), and disrupted by sonication. Cell lysates were cleared by centrifugation and desalted by repeated ultrafiltration in 20 mM Na-phosphate, pH 7.0, 0.5 mM dithiothreitol.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of Pea Stachyose Synthase-- Pea stachyose synthase was purified 619-fold from developing seeds by several chromatographic steps combined with preparative electrophoresis under nondenaturing conditions (Table I). After the last purification step on ceramic hydroxyapatite, 195 µg of protein were recovered from 280 g of seeds with a yield of 1.5% of the initial stachyose synthase activity. SDS-PAGE analysis of the purified protein revealed an apparent molecular mass of ~95 kDa (Fig. 1A). The protein was electroblotted and subjected to Edman degradation that yielded a single sequence of 25 amino acid residues, LIKTESIFDLSERKFKVKGFPLFHD. Western blot analysis (Fig. 1B) revealed cross-reactivity of the protein with polyclonal antibodies directed against stachyose synthase from adzuki bean seeds (21). All protein fractions obtained during the purification procedure were tested for their ability to catalyze the synthesis of verbascose in the presence of galactinol as donor or with stachyose as the sole substrate. Reaction products were separated and quantified by HPAEC-PAD. The ratio of stachyose synthase activity to the latter two activities remained almost constant throughout the purification procedure (Table I), suggesting that all reactions were catalyzed by a single protein.

View larger version (60K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE and Western blot analysis of purified pea stachyose synthase and lysates of insect cells expressing the recombinant protein. Proteins were separated by SDS-PAGE in 7.5% acrylamide gels and stained with silver nitrate (A) or subjected to Western blot analysis with polyclonal antibodies to adzuki bean stachyose synthase (B). Lane 1, purified pea stachyose synthase (0.2 µg); lane 2, soluble protein (2 µg) extracted from Sf21 insect cells expressing recombinant stachyose synthase; lane 3, uninfected Sf21 cells (control). M, molecular size markers. For Western blot analysis, prestained size markers were used.

Cloning and Expression of Stachyose Synthase cDNA in Insect Cells-- A PCR-based approach employing degenerate primers was used to isolate a cDNA fragment of ~1.9 kilobase pairs. Based on the sequence of this fragment, primers were designed to isolate the missing cDNA ends by ligation-mediated amplification. The assembled, contiguous cDNA sequence contains an open reading frame of 853 codons which encodes a protein of 95.9 kDa, in good agreement with the molecular mass of the purified protein. The amino-terminal sequence of the purified protein was located at positions 12 to 36 of the deduced amino acid sequence (Fig. 2). A sequence similarity search using the BLAST algorithm (23) revealed highest homology (75% identity and 83% similarity at the amino acid sequence level) to stachyose synthase from adzuki bean (21). Pea stachyose synthase also shows similarity with plant raffinose synthases (between 46 and 56%) and a family of plant seed imbibition proteins (SIP) of unknown function (between 28 and 30%). According to the classification system of Henrissat (24), all of these protein belong to glycosyl hydrolase family 36 of the glycosidase superfamily GH-D (for data base entries, see afmb.cnrs-mrs.fr/~pedro/CAZY/db.html). The positions of BLOCKS (25) glycoside hydrolase clan GH-D motifs (IPB000111) and a partial amino acid sequence alignment of pea stachyose synthase with members of the glycoside hydrolase family 36 are shown in Fig. 2. Motif IPB000111B is similar to the PROSITE (26) -galactosidase pattern PS00512. Motif IPB000111C is missing in stachyose synthases, raffinose synthases, and seed imbibition proteins. Instead, pea and adzuki bean stachyose synthase share a novel motif of about 80 amino acids (positions 304 to 383 of pea stachyose synthase). This block does not show significant homology to known sequences and, therefore, appears to be diagnostic for stachyose synthases.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Schematic representation of the amino acid sequence of pea stachyose synthase. The upper drawing depicts the primary structure of pea stachyose synthase. Positions of motifs of the BLOCKS (25) glycoside hydrolase clan GH-D signature (IPB000111) are indicated by capital letters. The shaded area depicts the location of a block specific for stachyose synthases. The position of the amino-terminal amino acid sequence of the purified protein is indicated by a black box. The lower panel shows a partial amino acid sequence alignment of pea stachyose synthase with members of glycoside hydrolase family 36. PsSTS, pea stachyose synthase; VaSTS, adzuki bean stachyose synthase; AtRFS, putative raffinose synthase from Arabidopsis thaliana; CsRFS, cucumber raffinose synthase; HvSIP, barley seed imbibition protein; BoSIP, wild cabbage seed imbibition protein; BbGAL, Bifidobacterium breve -galactosidase; TbGAL, Thermus brockianus -galactosidase. NCBI Protein Data base accession numbers are given in parentheses. Identical residues are represented by white letters on black background. Residues which are conserved in only a few sequences are shown on shaded background. Alignment was achieved with the program CLUSTAL W (39).

To confirm the substrate specificity of the purified enzyme, the open reading frame of the cDNA was amplified by PCR, cloned into a baculovirus expression vector, and expressed in insect cells under control of the polyhedrin promoter. Soluble proteins extracted from insect cells were separated by SDS-PAGE and subjected to Western blot analysis (Fig. 1B). In infected insect cells, a cross-reactive protein was detected at about 95 kDa, which did not occur in uninfected control cells. Crude, desalted extracts were assayed for enzymatic activity with various combinations of donor and acceptor substrates (Table II). Transferase activity was only observed with those substrates which were also utilized by the protein purified from pea seeds. No transferase activities was detected in uninfected control cells grown under identical conditions (data not shown). Collectively, these results demonstrate that the cloned cDNA encodes a functional stachyose synthase with a substrate specificity comparable to its natural, purified counterpart.

Substrate Specificity and pH Optimum-- A range of galactosyl acceptors were utilized by pea stachyose synthase when assayed in the presence of galactinol (Table II). Besides raffinose and stachyose, methylated inositol derivatives (D-ononitol, D-pinitol, and sequoyitol) were utilized, yielding galactosyl ononitol, galactopinitol A, and a galactosyl sequoyitol, respectively. The resulting galactosyl derivatives in turn could be utilized as donors for galactosyl transfer to raffinose and to stachyose, as shown for galactosyl ononitol in Table II. No transfer from galactinol to sucrose was observed, indicating that the protein is not able to initiate the biosynthesis of raffinose oligosaccharides. Likewise, no formation of stachyose was detected in the presence of raffinose as the sole substrate. When the enzyme was incubated with galactinol and verbascose, low amounts of a product were detected by HPAEC-PAD. Judged from its retention time, it appeared to be ajugose, the hexasaccharide of the raffinose series. No standard was available to confirm the identity of this reaction product. In addition to galactosyl transfer reactions, the purified protein was able to hydrolyze the terminal galactose residue of its substrates, although hydrolytic activities were generally low compared with the rate of the transfer reaction from galactinol to raffinose (Table II). Due to contaminating endogenous -galactosidase activity in lysates of insect cells, no attempts were made to determine the values for hydrolytic activities of the crude recombinant protein.

When assayed in Na-phosphate/citrate buffers, pH 4.0-8.5, the transfer reactions from galactinol to raffinose and to stachyose had an optimum at pH 7.0 (data not shown). Likewise, the rate of self-transfer from stachyose to stachyose was highest at pH 7.0, as previously shown for a crude enzyme preparation from pea seeds (16).

Steady-state Kinetics of Stachyose Synthesis and Product Inhibition by myo-Inositol-- The steady-state kinetics of stachyose synthesis were analyzed in the presence of nonsaturating concentrations of galactinol and raffinose (Fig. 3). A double reciprocal plot of the data yielded a set of parallel lines, suggesting that a double-displacement (ping-pong) catalytic mechanism was operative. In this mechanism, a putative glycosyl-enzyme intermediate is formed and the first product (myo-inositol) is released before the acceptor forms a complex with the enzyme. The initial velocity of the transfer reaction may thus be described by the initial rate equation for a double displacement mechanism,

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Dependence of the rate of stachyose synthesis on the concentrations of galactinol and raffinose. Double-reciprocal plot of stachyose synthase activity of purified pea stachyose synthase with respect to galactinol as the varied substrate. The concentrations of raffinose were 1.6 mM (), 2.7 mM (), 8 mM (), 16 mM (), or 40 mM (). Lines represent the best fit to Equation 1.

(Eq. 1)

where [Gol] and [Raf] are the concentrations of galactinol and raffinose, respectively, V is the maximum value of v, and K_Gol and K_Raf are Michaelis constants for galactinol and raffinose, respectively. Fitting the data from Fig. 3 to Equation 1 gave values of 13.9 ± 0.9 and 21.1 ± 1.3 mM for K_Gol and K_Raf, respectively. A value of 33.7 ± 1.1 nkat/mg of protein was obtained for V. Further support for a double-displacement mechanism came from product inhibition studies with myo-inositol. A double-reciprocal plot of the initial velocity versus the concentration of raffinose at several fixed concentrations of myo-inositol and a fixed subsaturating concentration of galactinol gave lines intersecting at the y axis, indicative of competitive inhibition with respect to raffinose (Fig. 4A). An apparent competitive inhibition constant of 2.3 ± 0.2 mM was obtained for myo-inositol as described elsewhere (27). Mixed inhibition was observed with galactinol as the variable substrate assayed at a fixed subsaturating concentration of raffinose (Fig. 4B), with apparent competitive and uncompetitive inhibition constants of 4.1 ± 0.6 and 9.2 ± 0.7 mM, respectively. This product inhibition pattern excludes reaction mechanisms involving ternary complexes (i.e. ordered and rapid equilibrium random mechanisms) (28).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Product inhibition of stachyose synthase activity by myo-inositol. A, double-reciprocal plot of inhibition of stachyose synthase activity by myo-inositol with respect to raffinose as the varied substrate. The concentration of galactinol was fixed at 10 mM. B, double-reciprocal plot of inhibition by myo-inositol with respect to galactinol as the varied substrate. The concentration of raffinose was fixed at 16 mM. The data were fitted by linear regression. The myo-inositol concentrations used were 0 mM (), 1 mM (), 5 mM (), or 10 mM ().

Steady-state Kinetics of Transfer Reactions Yielding Verbascose-- A double-reciprocal plot of the initial velocity of the transfer reaction from stachyose to stachyose versus the concentration of stachyose revealed a nonlinear pattern, while almost linear lines were obtained when galactinol was additionally present at fixed concentrations (Fig. 5A). Likewise, release of galactose (Fig. 5B) and raffinose (not shown) displayed complex, nonhyperbolic dependence on the concentration of the substrates. Under these conditions, transfer reactions from the two alternative donors as well as hydrolysis of the substrates occurred within the same time scale. A minimal double-displacement mechanism that accounts for the simultaneous occurrence of these reactions is shown in Scheme 1. Discrimination between competing substrates depends only on the concentration of the substrates and their relative V/K ratios (29). Thus, the ratio of the rate of release of raffinose (v_Raf) to that of myo-inositol (v_myo) is,

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Dependence of galactosyl transfer reactions to stachyose and hydrolysis of substrates on the concentrations of stachyose and galactinol. A, double-reciprocal plot of the initial velocity of formation of verbascose with respect to stachyose as the varied substrate. B, hydrolysis of substrates as determined by release of galactose with respect to stachyose as the varied substrate. C, plot of the ratio of rates of release of raffinose and myo-inositol versus the ratio of the concentrations of stachyose and galactinol. The rates of myo-inositol formation were calculated as described in the text. The data were fitted by linear regression (r2 = 0.998). The inset shows the data at low substrate ratios. D, plot of the ratio of formation of verbascose to that of galactose as a function of the concentration of stachyose. The data were fitted by linear regression (r2 = 0.986). In each case, the concentrations of galactinol were 0 mM (), 2 mM (), or 10 mM ().

View larger version (18K): [in this window] [in a new window]   Scheme 1.   Minimum double displacement mechanism for the synthesis of verbascose from stachyose and galactinol by pea stachyose synthase. Gal, galactose; Gol, galactinol; myo, myo-inositol; Raf, raffinose; St, stachyose; Ver, verbascose.

(Eq. 2)

where [St] and [Gol] are the concentrations of stachyose and galactinol, respectively, V_St and V_Gol are maximum velocities for galactosyl transfer from stachyose and galactinol, respectively, K_Gol is the Michaelis constant for galactinol and K_St is the corresponding constant for stachyose acting as donor. v_myo, which could not be determined with the HPAEC-PAD method used because of overlaping myo-inositol and galactinol peaks, was calculated as v_myo = v_Gal + v_Verb  v_Raf. A plot of v_Raf/v_myo versus the substrate ratio is shown in Fig. 5C. A straight line was obtained, confirming that galactinol and stachyose compete for the same active site. The V/K ratio for galactinol was ~9.5 times that for stachyose, indicating that galactinol was the preferred donor for the protein. The equation for partitioning of galactose units between stachyose and water as alternative acceptors may be derived from the initial rate equations (see Supplemental Materials) or directly by the net rate constant method (29),

(Eq. 3)

where k₇ is considered as pseudo-first order rate constant containing the water concentration factor. The ratio v_Ver/v_Gal is independent from the concentration of the donors. It is exclusively determined by competition between stachyose (as an acceptor) and water for galactose from a common intermediate, regardless whether this intermediate was formed from galactinol or from stachyose as the donor. A plot of v_Ver/v_Gal as a function of the concentration of stachyose is shown in Fig. 5D. Again, a straight line was obtained, as predicted by the proposed reaction pathway.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have isolated a stachyose synthase from developing pea seeds which catalyzed galactosyl transfer from galactinol to raffinose and to stachyose. The substrate specificity of the purified protein was confirmed by heterologous expression of the corresponding cDNA in insect cells. Our results corroborate the conclusions of Tanner and co-workers (11) published more than three decades ago, who suggested that a single transferase with a broad acceptor specificity could be responsible for chain elongation of raffinose in seeds of Vicia faba. Our results also demonstrate that there is a galactinol-independent pathway for the synthesis of verbascose in pea seeds. However, no evidence for the existence of a distinct GGT was found. Instead, stachyose synthase itself was found to be able to catalyze the synthesis of verbascose from stachyose as the sole substrate. Thus, the pathway of verbascose synthesis in pea seeds shows remarkable differences to that in leaf tissues, where the synthesis of stachyose takes place in the cytoplasm, while conversion to verbascose seems to be catalyzed by an acidic, vacuolar GGT (13, 14).

Steady-state kinetic data of the stachyose synthesis reaction (Fig. 3) and product inhibition by myo-inositol (Fig. 4) were in agreement with a double-displacement catalytic mechanism. Similar kinetic data have been reported for stachyose synthase from adzuki bean (12) and lentil (30). To date, no crystal structure is available and the predicted intermediary complex has not yet been characterized. However, isotopic exchange between galactinol and myo-inositol and between stachyose and raffinose has been demonstrated for a number of stachyose synthase preparations, providing good evidence for the proposed mechanism (12, 31, 32). Analysis of the reactions involving stachyose as a substrate was complicated by the dual role of stachyose as donor and acceptor and by simultaneous hydrolysis of substrates (Fig. 5, A and B). Nonetheless, discrimination of the enzyme between galactinol and stachyose as donors indicated that both donors compete for the same active site (Fig. 5C) and partitioning of galactose units between stachyose and water as alternative acceptors was compatible with the existence of a common intermediary complex (Fig. 5D). Then, self-transfer from stachyose simply represents the combination of reversal of the second half-reaction of stachyose synthase activity with the second half-reaction of verbascose synthase activity. The ratio V/K for stachyose as a donor was considerably lower than that for galactinol, suggesting that the contribution of the galactinol-independent pathway to the overall synthesis of verbascose in vivo is low unless the ratio of stachyose to galactinol is very high, which is only observed during late stages of seed development (16). It should also be noted that the water content of pea seeds progressively declines toward maturation, reaching a final value of as little as 0.1 g/g dry matter. Concomitantly, the concentration of raffinose oligosaccharides increases, on a plant water basis, dramatically. Under these conditions, the rates of hydrolysis of galactinol and raffinose oligosaccharides by stachyose synthase may be much lower compared with activities determined in aqueous solution.

A double-displacement mechanism also provides an explanation why the enzyme was able to catalyze rapid galactosyl transfer from galactinol to methylated derivatives of myo-inositol (Table II, Ref. 12). myo-Inositol is released before binding of the acceptor, making its binding site accessible for structural analogues during a catalytic cycle. Methylated inositols such as D-pinitol are not present in pea plants, but they are synthesized in other crops, such as soybean and lentil, and the corresponding -galactosides indeed accumulate in their seeds (9). Our results demonstrate that the absence of galactopinitols and related galactosyl inositols in pea seeds is caused by a lack of the substrates, and not by the absence of the enzymatic machinery required for their biosynthesis. Conversely, the low verbascose content of adzuki bean seeds (33) is in agreement with undetectable verbascose synthase activity of adzuki bean stachyose synthase (12). Subtle changes in the structure of the protein appear to be sufficient to eliminate the ability to utilize stachyose as an acceptor.

Stachyose synthases and raffinose synthases group into the glycoside hydrolase superfamily GH-D according to the sequence-based classification of Henrissat et al. (24). This clan of proteins comprises -galactosidases and -N-acetylgalactosaminidases from pro- and eukaryotes. Thus, from their primary structures, stachyose synthases and related enzymes should be classified as transglycosidases rather than as glycosyltransferases. The observation that stachyose synthases display at least weak hydrolytic activity (32, 34), while some -galactosidases display transglycosidase activity (35, 36), supports this classification. For -galactosidases of family 27, direct evidence has been presented that these glycosidases employ a double-displacement mechanism, where a side chain carboxylate acts as catalytic nucleophile to generate a glycosyl-enzyme intermediate (37, 38). Breakdown of the intermediate is presumably effected by a second carboxylate acting as acid-base catalyst. Relatively stable glycosyl-enzyme intermediates have been successfully trapped by using fluorosugars as substrates, allowing to identify an aspartic acid residue as the catalytic nucleophile (37, 38). It should be possible to use the fluorosugars developed for -galactosidases or similar active site labels to trap the proposed intermediate of stachyose synthases. On the other hand, the results presented here may also provide new starting points to explore structure-function relationships of -galactosidases.

	ACKNOWLEDGEMENT

We thank Barbara Svoboda for transfection and propagation of the insect cell cultures.

	FOOTNOTES

^* This work was supported by Austrian Science Fund (FWF) Grant P13955-BIO (to A. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Equations S1S6 and Table SI.

The nucleotide sequence(s) reported in this paper for pea stachyose synthase has been deposited in the GenBank^TM/EBI Data Bank with accession number(s) AJ311087.

^¶ To whom correspondence should be addressed: Chemical Physiology of Plants, Institute of Ecology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria. Tel.: 43-1-4277-54252; Fax: 43-1-4277-9542; E-mail: Andreas.Richter@univie.ac.at.

Published, JBC Papers in Press, October 23, 2001, DOI 10.1074/jbc.M109734200

	ABBREVIATIONS

The abbreviations used are: GGT, galactan:galactan galactosyltransferase; bis-Tris, 2-[bis(2-hydroxymethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; HPAEC-PAD, high performance liquid chromatography with pulsed amperometric detection.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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