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Plant Physiol. (1998) 117: 165-172
Galactosylononitol and Stachyose Synthesis
in Seeds of Adzuki
Bean1
Purification and Characterization of Stachyose Synthase
Thomas Peterbauer and
Andreas Richter*
Institute of Plant Physiology, University of Vienna, A-1091 Vienna,
Austria
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ABSTRACT |
Stachyose synthase (STS) (EC 2.4.1.67) was purified to homogeneity
from mature seeds of adzuki bean (Vigna angularis).
Electrophoresis under denaturing conditions revealed a single
polypeptide of 90 kD. Size-exclusion chromatography of the purified
enzyme yielded two activity peaks with apparent molecular masses of 110 and 283 kD. By isoelectric focusing and chromatofocusing the protein
was separated into several active forms with isoelectric point values between pH 4.7 and 5.0. Purified STS catalyzed the transfer of the
galactosyl group from galactinol to raffinose and
myo-inositol. Additionally, the enzyme catalyzed the
galactinol-dependent synthesis of galactosylononitol from
d-ononitol. The synthesis of a galactosylcyclitol by STS is
a new oberservation. Mutual competitive inhibition was observed when
the enzyme was incubated with both substrates (raffinose and ononitol)
simultaneously. Galactosylononitol could also substitute for galactinol
in the synthesis of stachyose from raffinose. Although galactosylononitol was the less-efficient donor, the Michaelis constant
value for raffinose was lower in the presence of galactosylononitol (13.2 mm) compared with that obtained in the presence of
galactinol (38.6 mm). Our results indicate that STS
catalyzes the biosynthesis of galactosylononitol, but may also mediate
a redistribution of galactosyl residues from galactosylononitol to
stachyose.
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INTRODUCTION |
RFO (e.g. raffinose,
stachyose, and verbascose) are among the most widespread soluble
 -galactosides that are accumulated during the development and
maturation of seeds (Dey, 1985 ). In plants containing cyclitols, such
as the methylated inositols ononitol
(1d-4-O-methyl-myo-inositol) and
pinitol
(1d-3-O-methyl-chiro-inositol), several nonreducing galactosylcylitols occur in addition to RFO. Galactosylcyclitols are frequently found in similar or even higher amounts than RFO in seeds of many important grain legumes, such as
lentil, chickpea, and soybean (Quemener and Brillouet, 1983; Horbowicz
and Obendorf, 1994). Both families of soluble -galactosides share
some common functions. They are generally regarded as being reserve
carbohydrates for the germinating seedling and have been proposed to
participate in the acquisition of desiccation tolerance and in the
viability of seeds (Horbowicz and Obendorf, 1994). The occurrence of
galactosides of methylated inositols is restricted to seeds,
whereas RFO are also found in storage tubers and leaves, and serve as
transport carbohydrates in the phloem (Kandler and Hopf, 1980).
RFO are synthesized by a set of distinct galactosyltransferases, which
sequentially add Gal units to Suc, yielding raffinose (raffinose
synthase, EC 2.4.1.82), stachyose (STS, EC 2.4.1.67), and higher
homologs (Kandler and Hopf, 1980, 1984). The galactosyl donor for these
reactions is galactinol
(O--d-galactopyranosyl-[1 1]-l-myo-inositol), which in turn is synthesized from myo-inositol and UDP-Gal
by the enzyme galactinol synthase (EC 2.4.1.123). This enzyme was characterized from cotyledons of bean (Liu et al., 1995) and other plant sources (Handley and Pharr, 1982; Webb, 1982; Smith et al., 1991). Considerably less is known about raffinose synthase, which has
only been examined as a partially purified preparation from broad bean
seeds (Lehle and Tanner, 1973). STS was originally described in seeds
of bean (Tanner and Kandler, 1968) but has only been purified to
homogeneity from leaves of melon (Holthaus and Schmitz,
1991).
Although the structure and distribution of many galactosylcyclitols are
well documented (for review, see Obendorf, 1997), very little
information is available on the biochemistry of these galactosides. We
have chosen ononitol
(1d-4-O-methyl-myo-inositol) and
galactosylononitol
(O--d-galactopyranosyl-[13]-4-O-methyl-d-myo-inositol) as a model system for the characterization of galactosylcyclitol biosynthetic pathways (Richter et al., 1997). In an initial study we
identified a galactosyltransferase activity in extracts of seeds of
adzuki bean (Vigna angularis) that utilizes
galactinol in the biosynthesis of galactosylononitol (Peterbauer and
Richter, 1997). By analogy with similar galactosyltransferases, we have termed this activity GOS (Fig. 1). GOS
and STS activity copurified with a constant activity ratio during
several chromatographic steps, suggesting that
galactosylononitol synthesis may be catalyzed by STS. The present study
therefore was aimed at clarifying whether STS participates in
galactosylononitol metabolism. We report on the purification and
characterization of the enzyme from seeds of V. angularis,
and demonstrate that STS is a multisubstrate enzyme that is able to
synthesize galactosylononitol (GOS activity) in addition to stachyose
(STS activity).
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| Figure 1.
Reaction scheme of GOS activity. The galactosyl
moiety of galactinol is transfer red to d-ononitol,
yielding galactosylononitol and myo-inositol. The
reaction is reversible.
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MATERIALS AND METHODS |
Plant Material and Chemicals
Seeds from adzuki bean (Vigna angularis [Willd.] Ohwi
& Ohashi) were obtained from a local market. Galactinol was purified from leaves of sage (Salvia officinalis) as previously
described (Kuo, 1992). Ononitol and galactosylononitol were isolated
from seeds of V. angularis as previously described (Richter
et al., 1997). Further substrates (d-pinitol, sequoyitol,
d- and l-bornesitol, l-quebrachitol,
1-O-methyl-scyllo-inositol, d- and
l-chiro-inositol, d-1-O-methyl-muco-inositol, and
muco-inositol) were isolated and purified as previously
described (Wanek and Richter, 1995). All other chemicals were obtained
from commercial sources and were of the highest purity available.
Enzyme Purification
Preparation of Extract
Seeds were frozen in liquid N2 and ground to
a fine powder in a sample mill (IKA A10, Janke and Kunkel, Germany).
Approximately 125 g of the powder was suspended in 480 mL of
ice-cold extraction buffer (100 mm Hepes-NaOH, pH 7.0, 1 mm DTT, 1 mm EGTA, and 20 mm
MgCl2) that contained 5 g of
polyvinylpolypyrrolidone, and was further homogenized with a Polytron
tissue homogenizer. The suspension was filtered through fine-mesh nylon
(42 µm) and centrifuged at 26,000g at 4°C for 30 min.
The supernatant was used for further protein purification.
Protamine Sulfate and Ammonium Sulfate Precipitation
All manipulations were carried out at 4°C. A 10% (w/v)
protamine sulfate solution (in extraction buffer) was slowly added to
the crude extract to a final concentration of 2 g
L 1. Precipitated protein was removed by
centrifugation (30 min at 26,000g) and the supernatant was
fractionated with solid ammonium sulfate. Proteins precipitating
between 35 and 55% saturation were collected by centrifugation for 20 min at 26,000g.
HIC
The pellet of the ammonium sulfate fractionation was dissolved in
HIC buffer (50 mm Hepes-NaOH, pH 7.0, 1 mm DTT,
and 1 m ammonium sulfate) and loaded onto a 70-mL column of
Phenyl Sepharose HP (Pharmacia) preequilibrated in the same buffer at
20°C. Bound protein was eluted by applying a linear gradient of 1.0 to 0.0 m ammonium sulfate in HIC buffer (700 mL) at a
constant flow rate of 5 mL min1.
Fifteen-milliliter fractions were collected and assayed for STS
activity. Active fractions were pooled and concentrated by repeated
ultrafiltration (Ultrafree-15, Biomax 10K, Millipore) in PAGE sample
buffer (125 mm Tris-HCl, pH 6.8, containing 1 mm DTT, 10% [v/v] glycerol, and 0.0015% [w/v]
bromphenol blue) at 4°C.
Preparative Native PAGE
Preparative PAGE was performed under nondenaturating conditions on
a Prep Cell (model 491, Bio-Rad). A 7% acrylamide resolving gel (7 cm
high) and a 3.75% acrylamide stacking gel (2 cm high) were cast in a
37-mm i.d. gel tube in 123 mm bis-Tris-HCl (pH 6.61). The
upper running buffer consisted of 44 mm Tes and 113 mm bis-Tris (pH 7.25). The lower running buffer (63 mm bis-Tris-HCl, pH 5.9) was cooled by circulation through
a water bath (4°C). The sample of the HIC step (3 mL) was
electrophoresed at 12 W of constant power. Proteins were eluted with
bis-Tris-HCl (113 mm, pH 7.0, 1 mm DTT,
and 10% [v/v] glycerol) at a flow rate of 0.8 mL
min1, and 4-mL fractions were collected. Every
second fraction was assayed for STS activity.
AEC
Active PAGE fractions were pooled, desalted, and
concentrated at 4°C by repeated ultrafiltration in AEC sample buffer
(20 mm bis-Tris-HCl, pH 7.0, 1 mm DTT and 10%
[v/v] glycerol) as described above. The sample was applied at 2 mL
min1 to an anion-exchange column (6-mL bed
volume, Resource Q, Pharmacia,) preequilibrated in AEC sample buffer.
Bound protein was eluted with 90 mL of a linear gradient of 0.0 to 0.3 m NaCl in AEC sample buffer. Fractions (1.5 mL each) that
contained STS activity were pooled, concentrated at 4°C by
ultrafiltration (Centricon-10, Amicon, Beverly, MA), and stored in
liquid N2.
Enzyme and Protein Assay
STS activity was routinely determined in reaction mixtures that
contained 50 mm Hepes-NaOH (pH 7.0), 1 mm DTT,
10 mm galactinol, 50 mm raffinose, and 3 to 30 pkat of enzyme activity in a final volume of 60 µL. Assays were
incubated at 30°C for 30 min and terminated by boiling for 5 min.
After centrifugation, the supernatant was deionized by the use of
ion-exchange resins (Dowex 50-100 mesh: 50WX8,
H+-form; 1X8, formate form; Sigma). The formation of
stachyose was monitored by HPLC with pulsed amperometric detection (DX
500, Dionex, Sunnyvale, CA) on a Carbopac PA10 column (250 × 4 mm, Dionex) with 100 mm NaOH as the eluent at a flow rate
of 1 mL min1 (30°C).
For determination of GOS activity, enzyme preparations were incubated
with 20 mm d-ononitol and 10 mm
galactinol. Reaction products were separated by HPLC with pulsed
amperometric detection on a Carbopac MA1 column (250 × 4 mm,
Dionex) with 100 mm NaOH at a flow rate of 0.4 mL
min1 at 20°C. The identity of reaction
products was checked by GC-MS (see below).
Protein concentrations were estimated with BSA as a standard, using the
Bradford dye-binding procedure (protein assay, Bio-Rad).
Enzyme Characterization
Kinetic Analysis and Substrate Specificity
Km and
Vmax values were obtained from slope and
intercept replots of initial rate data as described by Rudolph and
Fromm (1979). The concentrations of substrates were varied as follows:
raffinose from 5.5 to 50 mm at (a) 1.1 to 10 mm
galactinol or (b) 2.2 to 20 mm galactosylononitol, and
d-ononitol from 2.2 to 20 mm at 1.1 to 10 mm galactinol. Kinetic values estimated at only one concentration of the fixed substrate are referred to as
Km(app) and
Vmax(app) values, respectively. Inhibition
constants were calculated from replots of the slopes from primary
double-reciprocal plots against the inhibitor concentration.
The reaction products of substrate-specificity assays were
analyzed by GC-MS (Richter, 1992). Deionized assays were taken to
dryness and the reaction products were converted to trimethylsilyl derivatives by treatment with
pyridine: N,O-bis-(trimethylsilyl)-trifluoroacetimide:trimethyl-chlorosilane (40:10:1; v/v) at 75°C for 60 min. The trimethylsilyl
derivatives were separated on a fused silica column (DB5-ms,
30 m in length, 0.25 mm i.d., and 0.1-µm film thickness; J & W
Scientific, Folsom, CA) with He as the carrier gas at 140 kPa
column-head pressure. The oven temperature was programmed
from 110°C (1 min) to 320°C at 8°C min1.
Mass spectral data were obtained with an ion-trap mass
spectrometer (Saturn 3, Varian, Sugarland, TX) at a source
temperature of 260°C in the electron-impact-ionization mode.
SDS-PAGE and Size-Exclusion Chromatography
Discontinuous SDS-PAGE was carried out under reducing conditions
using 8 to 18% precast gradient gels (Excel Gel SDS, Pharmacia), according to the manufacturer s recommendations, and calibrated with
SDS molecular-mass standards in the range of 14.4 to 97.4 kD (Bio-Rad).
Proteins were visualized by silver staining. The molecular mass of
native STS was estimated by using size-exclusion chromatography. A
0.1-mL sample of purified enzyme was applied at 0.5 mL
min1 to a Superdex 200 HR 10/30 column (Pharmacia)
pre-equilibrated in 20 mm NaPi (pH 7.0), 150 mm
NaCl, and 0.5 mm DTT. Fractions of 0.25 mL were collected
and assayed for STS and GOS activity. The column was calibrated with
reference proteins of known molecular masses (18-300 kD,
Combithek, Boehringer Mannheim).
IEF and Chromatofocusing
IEF was performed on Ampholine gels (pH 3.5-9.5, Pharmacia),
calibrated with standard proteins (IEF-mix, pI 3.6-9.3, Sigma). Proteins were visualized by silver staining. For chromatofocusing, a
sample of purified enzyme was diluted with 25 mm
bis-Tris-HCl (pH 6.3) containing 0.5 mm DTT, concentrated
by ultrafiltration (Centricon-10, Amicon), and applied at 1 mL
min1 to a Mono P HR 5/5 column (Pharmacia)
pre-equilibrated in the same buffer. The column was eluted with a pH
gradient (6.3-4.0) formed by 10% (v/v) Polybuffer 74 (Pharmacia)
containing 0.5 mm DTT. Fractions of 0.25 mL were collected,
and an equal volume of 0.5 m NaPi (pH 7.0, 1 mm
DTT) was added. Fractions were desalted and concentrated by repeated
ultrafiltration (Centricon-10, Amicon) in 20 mm NaPi (pH
7.0) and 1 mm DTT at 10°C, and assayed for STS and GOS
activity.
N-Terminal Amino Acid Sequencing
A sample of STS was subjected to SDS-PAGE as described above and
electroblotted onto a PVDF membrane (Bio-Rad) with 10 mm 3-(cyclohexylamino)-1-propane-sulfonic acid (pH 11.0) containing 10%
(v/v) methanol. The membrane was stained with Coomassie blue R-250, the
band at 90 kD was cut out, and the N-terminal amino acid sequence was
analyzed at the Institute of Biochemistry (University of Vienna) using
a sequencer (model 476A, Applied Biosystems).
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RESULTS |
Purification of STS
STS was purified 244-fold from mature seeds of V. angularis. The results of a typical purification procedure are
summarized in Table I. An initial
protamine sulfate and ammonium sulfate fractionation removed lipid
contaminants and 38% of the protein from the crude extract. HIC on
Phenyl Sepharose HP (Fig. 2A) provided a
significant purification and substantially reduced the total amount of
protein. The key step in the purification procedure was native
preparative PAGE at neutral pH (Fig. 2B). By using this technique STS
was resolved highly purified and with an excellent yield of activity
(Fig. 2B). Remaining contaminants were removed by AEC on a Resource Q
column (Fig. 2C), yielding a 244-fold purification with a recovery of
7.9% (Table I). The final preparation had a specific STS activity of
11.2 nkat mg1 protein and was apparently
homogenous by SDS-PAGE (Fig. 3A). N-terminal sequencing of the purified protein revealed a single sequence of 24 amino acids (NDPVNATLGLEPsEKVFDLLDGKL).
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Table I.
Purification of STS from mature seeds of V. angularis
Enzyme activity was assayed with 10 mm galactinol and 50 mm raffinose.
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| Figure 2.
Purification of STS from seeds of V. angularis. A, HIC of proteins on Phenyl Sepharose HP after
protamine sulfate treatment and ammonium sulfate fractionation (dashed
line, 1.0-0.0 m ammonium sulfate). B, Native preparative
PAGE of pooled fractions from the HIC on a Bio-Rad PrepCell 491. C,
Final purification by AEC on Resource Q (dashed line, 0.0-0.3
m NaCl).
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| Figure 3.
Analysis of STS at various stages of purification.
A, SDS-PAGE. Lane 1, Crude protein extract; lane 2, 35 to 55% ammonium sulfate precipitate after protamine sulfate treatment; lane 3, pooled
fractions after chromatography on Phenyl Sepharose HP; lane 4, molecular mass markers; lane 5, pooled fractions after native PAGE; and
lane 6, pooled fractions after chromatography on Resource Q. B, IEF of
purified STS. Lanes were loaded with 0.1 to 1.0 µg of protein and
stained with silver nitrate.
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Physicochemical Properties and Effectors
Purified STS was subjected to size-exclusion chromatography on a
Superdex 200 HR column for determination of the native molecular mass.
A minor and a major peak of 283 and 110 kD, respectively, were observed
(Fig. 4A). The 110-kD protein was
isolated and reinjected onto the size-exclusion column. Again a 283-kD
peak was observed in addition to the 110-kD peak, indicating that the
enzyme forms aggregates under the conditions used. On SDS-PAGE gels
both the 110- and 283-kD forms exhibited a single band corresponding to a molecular mass of 90.1 kD (Fig. 3A).
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| Figure 4.
Size-exclusion chromatography (A) and
chromatofocusing (B) of purified STS. STS ( ) and GOS ( )
activities were determined by HPLC as described in ``Materials and Methods''. A, Elution profile from a Superdex 200 HR 10/30 column.
Inset, Native molecular mass estimation. The apparent molecular masses
of STS (110 and 283 kD) are indicated by arrows. B, Elution profile
from a Mono P HR 5/5 column. The pH gradient ( ) was formed with
Polybuffer 74.
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Purified STS was subjected to IEF under native conditions, yielding
several bands in the range of pI 4.7 to 5.0 (Fig. 3B). By
chromatofocusing on a Mono P column, several unresolved active peaks
were observed in a similar pH range (Fig. 4B), possibly due to
aggregate formation. Since the enzyme was unstable at low pH values, no
attempts were made to get further insight into the observed
microheterogeneity.
STS exhibited a temperature optimum at around 35°C and a
one-half-maximum activity at approximately 23°C (Fig.
5A). The pH-dependent activity curve
showed a maximum between pH 6.5 and 7.0 in McIlvaine buffer
(NaPi-citrate) (Fig. 5B). STS activity did not require cations, but was
strongly inhibited by Mn2+,
Zn2+, Cu2+, and
Fe2+ (Table II).
The presence of DTT slightly increased the activity, although 20 µm PCMBS had only a minimal effect on the enzyme (Table II). The purified enzyme was stable for at least 4 months when stored
in liquid N2. When stored at  20°C, 85% of
activity was lost within 4 months.
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| Figure 5.
Effect of temperature (A) and pH (B) on STS ()
and GOS () activity. A, Temperature-dependent activity profiles
assayed in 50 mm Hepes-NaOH (pH 7.0) and 1 mm
DTT. Data were adjusted relative to the maximum activity measured. B,
pH-dependent activity profiles assayed in McIlvaine buffer and 1 mm DTT at 30°C. Data were adjusted relative to the
maximum activity measured.
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Table II.
Influence of cations and other agents on STS
activity from V. angularis
The enzyme activity was assayed with 10 mm galactinol, 50 mm raffinose, and the indicated additions. Activity of the
control (no addition) was 25 pkat per assay.
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Reactions and Kinetics
The purified enzyme catalyzed several galactosyl transfer
reactions (Table III). For the genuine
STS activity,
Km values of 15.8 and 38.6 mm were estimated at nonsaturating substrate levels for
galactinol and raffinose, respectively, with a
Vmax of 77.5 nkat
mg1 protein (Table
IV). Apparent affinities
(Km[app] values) for both substrates were
markedly affected by the concentration of the respective fixed
cosubstrate. In the presence of the lowest concentration of galactinol
used (1.1 mm), a Km(app) value
of 2.4 mm was found for raffinose, whereas a
Km(app) of 1.8 mm was found for
galactinol in the presence of 5.5 mm raffinose.
myo-Inositol acted as a competitive inhibitor relative to
raffinose with a Ki of 4.6 mm
(data not shown). As already described for STS from other plant sources
(Tanner and Kandler, 1968; Gaudreault and Webb, 1981), the enzyme from
V. angularis also catalyzed the following exchange reaction:
When assayed with 10 mm galactinol, a
Km(app) of 5.2 mm was estimated
for myo-[3H]inositol. However, the
enzyme was not specific for galactinol and myo-inositol, but
also catalyzed the following reaction, in a manner similar to that of
reaction 1:
Thus, the enzyme accepted galactosylononitol instead of galactinol
as a galactosyl donor, indicating an alternative biosynthetic pathway
for stachyose. A fairly high Km value was
obtained for galactosylononitol (31.3 mm) with a
Vmax of 46.2 nkat
mg1, whereas the Km
value for raffinose (13.2 mm) was considerably lower in the
presence of galactosylononitol instead of galactinol (Table IV). In
addition, the purified enzyme was capable of catalyzing the synthesis
of galactosylononitol from galactinol and ononitol (GOS activity, Fig.
1):
This reaction closely resembles (exchange) reaction 2, with
ononitol substituting for
myo-[3H]inositol. For the GOS
reaction, Km values were estimated to be
6.3 and 18.1 mm for galactinol and ononitol, respectively
(Table IV). The reaction was readily reversible. A rate of 21.6 nkat mg1 was observed in the presence of 10 mm galactosylononitol and 20 mm
myo-inositol.
STS and GOS activities exhibited identical temperatures and pH optima
(Fig. 5, A and B) and were catalyzed by all forms of the enzyme
separated by size-exclusion chromatography (Fig. 4A) and
chromatofocusing (Fig. 4B), although STS activity appeared to be less
stable than GOS activity at low pH values. Simultaneous incubation of
the enzyme with galactinol and both acceptors (ononitol and raffinose)
resulted in mutual inhibition (Fig. 6).
Ononitol acted as a competitive inhibitor toward raffinose, whereas
raffinose acted as a competitive inhibitor with respect to
ononitol. Apparent Ki values of 18.7 and 17.9 mm for ononitol and raffinose, respectively, were estimated from replots of slopes (Fig. 6).
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| Figure 6.
Mutual inhibition of STS and GOS activity. The
reaction mixtures contained 10 mm galactinol, 0 to 50 mm raffinose, and 0 to 20 mm ononitol. Assays
were analyzed for both stachyose and galactosylononitol. Data were
plotted as follows: A, Formation of stachyose at different concentrations of ononitol with raffinose as the variable substrate. Ononitol concentrations were: 1, 0 mm; 2, 2.9 mm; 3, 4 mm; 4, 6.7 mm; and 5, 20 mm. B, Formation of galactosylononitol at different concentrations of raffinose with ononitol as the variable substrate. The raffinose concentrations were: 1, 0 mm; 2, 7.1 mm; 3, 10 mm; 4, 16.7 mm; and 5, 50 mm.
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Several isomeric inositols and inositol
O-methyl-ethers were tested as possible galactosyl acceptors
(Table III). Under the conditions used, myo-inositol was the
most effective acceptor (224% compared with raffinose). Methylation or
epimerization of the hydroxyl groups at carbons C-2, C-4, C-5, and C-6
of the myo-inositol ring yielded the derivatives
d-ononitol, sequoyitol, scyllo-inositol, O-methyl-scyllo-inositol, and
epi-inositol, respectively, which were utilized at rates
between 4 and 127%. Those derivatives of myo-inositol, that
are modified at C-1 or C-3 (d- and
l-bornesitol, d- and
l-chiro-inositol, l-quebrachitol,
muco-inositol, and
d-1-O-methyl-muco-inositol) were found to be inactive in the system used. d-Pinitol, a
naturally occurring cyclitol in many legumes, was only accepted at a
very low rate. All attempts to demonstrate a galactosyl transfer from galactinol to Suc or to stachyose were not successful (Table III).
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DISCUSSION |
Although STS has previously been characterized in enzyme
preparations from several different plant species and has been purified to homogeneity from leaves of melon (Holthaus and Schmitz, 1991), this
is the first report to our knowledge on the purification of the enzyme
to homogeneity from a seed source. STS from V. angularis exhibited a broad pH optimum between pH 6.5 and 7.0 and a temperature optimum at around 35°C, similar to that of the partially purified enzyme from seeds of bean (Tanner and Kandler, 1968) and that of leaves
of squash (Gaudreault and Webb, 1981), melon (Holthaus and Schmitz,
1991), and common bugle (Bachmann et al., 1994). However, the enzyme
from seeds of V. angularis differed markedly from that of
melon leaves with respect to molecular mass. The leaf enzyme was
reported to consist of two subunits of 45 and 50 kD (Holthaus and
Schmitz, 1991). In contrast, SDS-PAGE of the enzyme from V. angularis seeds revealed a single polypeptide of 90.1 kD (Fig.
3A). No native molecular mass data and amino acid sequence information
are available for the purpose of a comparison.
The enzyme from V. angularis catalyzed an exchange reaction
between galactinol and (labeled) myo-inositol at a rate
comparable to that reported for preparations from bean and melon
(Tanner and Kandler, 1968; Gaudreault and Webb, 1981). For this
galactosyl exchange, a reaction mechanism has been proposed in which
galactinol reacts with the enzyme to form a Gal-enzyme complex and
myo-inositol that dissociates, making way for (labeled)
myo-inositol (Tanner and Kandler, 1968; Dey, 1985). Several
other cyclitols could substitute for myo-inositol in the
exchange reaction catalyzed by the purified V. angularis
enzyme, resulting in a net synthesis of galactosylcyclitols (Table
III). The reversible synthesis of galactosylononitol from galactinol
and d-ononitol (Fig. 1) deserves special attention, since
ononitol is the only naturally occurring O-methyl-inositol in V. angularis and its galactosyl derivative is accumulated
in seeds of many legumes (Yasui et al., 1985, 1987; Obendorf, 1997). To
our knowledge, this is the only known route for galactosylononitol biosynthesis. The catalytic efficiency for galactosylation of ononitol
(calculated as
Vmax/Km) was
almost 1.9-fold higher compared with that of raffinose (both assayed
with galactinol), providing evidence that this pathway is active in
vivo (Table IV). It is interesting that galactopinitols, although
widespread in legume seeds, were not synthesized by STS from V. angularis to a significant extent.
The involvement of galactinol as a cofactor in the biosynthesis of RFO
is firmly established by in vitro and in vivo studies (Senser and
Kandler, 1967; Kandler and Hopf, 1980). In addition, purified STS from
V. angularis also accepted galactosylononitol as a
galactosyl donor in the formation of stachyose, but with a lower
catalytic efficiency (Table IV). Although already proposed by others
(Beveridge et al., 1977; Dey, 1985), our results provide the first
evidence to our knowledge that a galactoside of an inositol O-methyl-ether is involved in the RFO metabolism of a legume
seed. We were able to demonstrate that ononitol is a product (arising from galactosylononitol-dependent synthesis of stachyose) and a
substrate for the enzyme (GOS activity). Most likely, both reactions are catalyzed at one active site, in agreement with the observed competitive inhibition by myo-inositol and ononitol versus
raffinose (Fig. 6), as well as with the proposed mechanism of the
above-described exchange reaction.
The enzyme from V. angularis seeds displayed low affinity
toward raffinose when assayed with galactinol
(Km of 38.6 mm), compared with
melon STS (Km between 3.7 and 15 mm) (Huber et al., 1990; Holthaus and Schmitz, 1991).
However, the Km value for raffinose was
markedly lower (13.2 mm) for STS of V. angularis
when assayed with galactosylononitol instead of galactinol (Table IV).
It is interesting that the concentration of galactosylononitol in
developing V. angularis seeds was consistently higher than
that of galactinol and raffinose throughout stachyose accumulation (T. Peterbauer, M. Puschenreiter, and A. Richter, unpublished results),
suggesting that galactosyl transfer from galactosylononitol to
raffinose may significantly contribute to the formation of stachyose in vivo. Nevertheless, since galactinol is the only known galactosyl donor
in the formation of galactosylononitol, the biosynthesis of stachyose
via galactosylononitol ultimately seems to depend also on
galactinol.
Galactosylcyclitols accumulate alongside RFO during the acquisition of
dessication tolerance in legume seeds (Obendorf, 1997). However, the
physiological significance of galactosylononitol in seeds is not fully
understood at present. We have demonstrated here that the biosynthesis
and metabolism of galactosylononitol and stachyose are linked via the
enzyme STS in V. angularis seeds. It may therefore be
possible that more enzymes are shared by the metabolic pathways of
galactosylcyclitols and RFO. However, inhibitory effects of cyclitols
on enzymes of RFO synthesis cannot be excluded. A reinvestigation of
the RFO metabolic enzymes with respect to cyclitol and
galactosylcyclitol specificity is clearly needed.
| |
FOOTNOTES |
1
This work was supported by the Austrian Science
Foundation (project no. P10917-BIO).
*
Corresponding author; e-mail arichter{at}pflaphy.pph.univie.ac.at;
fax 43-1-31336-776.
Received November 5, 1997;
accepted January 27, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AEC, anion-exchange chromatography.
GOS, galactosylononitol synthase.
HIC, hydrophobic-interaction
chromatography.
RFO, raffinose family oligosaccharide(s).
STS, stachyose synthase.
| |
ACKNOWLEDGMENTS |
We wish to thank Prof. M. Popp and Dr. W. Wanek for valuable
comments on the manuscript and Dr. R. Prohaska for protein sequencing.
| |
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Top
Abstract
Introduction
![<IT>myo-</IT><UP>[<SUP>3</SUP>H]inositol+galactinol→[<SUP>3</SUP>H]galactinol</UP>](/content/vol117/issue1/fulltext/165/img002.gif)
