Plant Cell Physiol. 39(12): 1337-1341 (1998)
JSPP © 1998
An Increase in Apparent Affinity for Sucrose of Mung Bean Sucrose
Synthase Is Caused by In Vitro Phosphorylation or Directed Mutagenesis of
Ser11
Tomonori Nakai 1 , Teruko Konishi', Xiu-Qing Zhang 2 , Raymond Chollet2, Naoto Tonouchi 3 ,
Takayasu Tsuchida3, Fumihiro Yoshinaga3, Hitoshi Mori 4 , Fukumi Sakai1 and Takahisa Hayashi 1,5
' Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto, 611-0011 Japan
Department of Biochemistry, University of Nebraska-Lincoln, George W. Beadle Center, Lincoln, NE 68588-0664, U.S.A.
3
Bio-Polymer Research Co. Ltd., KSP, Takatsu-ku, Kawasaki, 213 Japan
4
Faculty of Agriculture, Nagoya University, Chikusa, Nagoya, 464-01 Japan
2
A mutational analysis of mung bean (Vigna radiata
Wilczek) sucrose synthase was performed by site-directed
mutagenesis of the recombinant protein expressed in Escherichia coli, in which two different acidic amino acid residues (Asp or Glu) were introduced at Ser11 (S11D, SHE).
Only the wild-type enzyme (Ser11) was phosphorylated in
vitro by a Ca 2+ -dependent protein kinase from soybean
root nodules, suggesting that this is the specific target residue in mung bean sucrose synthase. The apparent affinity
for sucrose was increased in this phosphorylated enzyme
and also in the S11D and S H E mutant enzymes, although
the affinities for UDP-glucose and fructose were similar in
the wild-type, phosphorylated wild-type, and mutant enzymes. These results suggest that a monoanionic (1~) side
chain at position 11 mimics the Ser"-P 2 ~ residue to bind
and cleave sucrose for the synthesis of UDP-glucose. Since
the S H E mutant enzyme showed the lowest Km (sucrose)
and the highest catalytic efficiency of the recombinant proteins, the enzymic properties of this S H E mutant were further characterized. The results showed that replacement of
Ser11 with Glu 11 modestly protected the sucrose synthesis activity against phenolic glycosides and altered the enzyme nucleotide specificity. We postulate that the introduction of
negative charge at Ser11 is possibly involved in the enzymatic perturbation of sucrose synthase.
Key words: Directed mutagenesis — Protein phosphorylation — Sucrose synthase — Vigna radiata Wilczek.
Sucrose synthase (EC 2.4.1.13) catalyzes the synthesis
and cleavage of sucrose: UDP-glucose + fructose ^ sucrose + UDP, a reversible reaction. The enzyme occurs predominantly in the non-photosynthetic "sink" tissues of
higher plants, where sucrose is the major form of carbon
Abbreviations: CDPK, Ca2+-dependent protein kinase;
DTIV dithiothreitol; FPLC, fast protein liquid chromatography;
HEPES, N-2-hydroxyethylpiperazine-/V-2-ethanesuIfonic acid.
5
Corresponding author: e-mail, [email protected]
that is translocated and cleaved subsequently by its activity
to produce UDP-glucose for the synthesis of cell walls or
starch (Chourey and Nelson 1976, Amor et al. 1995). This
production of UDP-glucose can be viewed as a form of
energy-saving mechanism for ATP in growing cells because
the UDP formed from UDP-glucose by glucosyltransferase
reactions can be efficiently and rapidly recycled to produce
UDP-glucose by sucrose synthase.
The activity of sucrose synthase is potentially regulated by reversible seryl-phosphorylation, which has been observed at Ser15 in the maize SS-2 isoform (Shaw et al. 1994,
Huber et al. 1996) and presumably at Ser" in soybean root
nodule sucrose synthase (Zhang and Chollet 1997, Zhang
et al. 1997). This phosphorylation event may activate the
formation of UDP-glucose and fructose from sucrose plus
UDP by increasing the apparent affinity of the enzyme for
sucrose and UDP (Huber et al. 1996, Winter et al. 1997).
Comparison of the amino acid consensus sequence revealed that sucrose synthases from several plant species have a
conserved Ser residue for phosphorylation in the related
phosphorylation motif, basic-x-x-Ser-hydrophobic (Huber
et al. 1996). We have recently reported that the recombinant sucrose synthase from mung bean (Vigna radiata
Wilczek) has a 10-fold lower apparent affinity for sucrose
but essentially the same affinities for UDP-glucose and fructose as that of the authentic plant enzyme (Nakai et al.
1997). In the present study, we have examined whether the
low apparent affinity for sucrose of this non-phosphorylated recombinant mung bean enzyme is increased not only
by in vitro phosphorylation but also by site-directed mutagenesis that replaces Ser" with either an anionic Asp
(SIID) or Glu (SHE) residue.
Materials and Methods
Bacterial strains and media—Escherichia coli strain BL21
(DE3) was used as the host to produce wild-type and mutant
recombinant sucrose synthases from mung bean by using the pET21d expression vector under the control of a T7 promoter
(Novagen). Transformants of strain BL21(DE3) containing various plasmids were cultivated as described previously (Nakai et al.
1997).
1337
1338
Affinity for sucrose of sucrose synthase
Construction of expression plasmids—Sucrose synthase
cDNA was used for amplification by PCR using plasmid pM-SS-5
as the template (Arai et al. 1992, Nakai et al. 1997). Oligonucleotide N-ter2 was used as primer for the wild-type (Ser") enzyme
and two synthetic oligonucleotides were used as primers for mutagenesis of Ser" to either Asp or Glu (Fig. 1). The PCR product
was digested with Sail and cloned into the pET-21d vector that
had been digested with Ncol, filled with Klenow fragment, and
then digested with Xhol. After ligation, the products were used to
transform E.coli strain BL21(DE3) and a mini-preparation was
used to isolate the recombinant plasmids (Ausubel et al. 1988).
The alignments of the resulting plasmids, designated as pED-01,
pED-01-Sl ID, and pED-01-Sl IE, respectively, were confirmed by
sequencing the entire cDN A insert according to the primer-labeled
dideoxy-chain-termination method (Sanger et al. 1977).
Purification of the recombinant sucrose synthases—Recombinant protein production was induced at 37°C by addition of isopropyl thio-yS-galactoside to the cell culture (3 liters) at a final concentration of 0.3 mM. After continued growth at 37°C for 3 h and
subsequent harvesting by centrifugation, the cells were resuspended in 30 mM Tris-HCl, pH7.5, containing 0.1 mM EDTA,
0.1 mM dithiothreitol (DTT), and disrupted by sonication on ice.
The broken cell suspension was centrifuged at 30,000 xg for 30
min and the crude supernatant fraction was recovered. To the supernatant fluid, ammonium sulfate was added to 30% saturation
and the resulting precipitate discarded. The corresponding supernatant fraction was brought to 65% saturation by further addition
of ammonium sulfate. The precipitate was then dissolved in 4 ml
of 30 mM Tris-HCl, pH 7.4. The solution was dialyzed against
500 ml of 30 mM Tris-HCl, pH 7.4, for 30 min. The clarified solution was then passed through a Sepharose CL-6B gel filtration column (5 x 20 cm [Pharmacia]) that had been pre-equilibrated with
the same buffer at 4°C. The flow rate was adjusted to 2 ml per min
with an FPLC system (Pharmacia), and 5-ml fractions were collected. The peak fractions of sucrose synthase activity from this
column were pooled and the protein was precipitated with 65% saturation of ammonium sulfate. The precipitate was resuspended in
4 ml of 30 mM Tris-HCl, pH7.5, containing 500 mM NaCl, 1
mM EDTA, and 1 mM DTT. The clarified solution was passed
through a Hi Load Superdex 200 size-exclusion column (2.6x60
cm [Pharmacia]) equilibrated with resuspension medium. The
flow rate of the buffer was adjusted to 2 ml per min and 5-ml fractions were collected. The peak activity fractions were pooled and
dialyzed overnight at 4°C against 3 liters of 30 mM Tris-HCl, pH
7.4. The desalted solution was placed on a prepacked Mono-Q
HR5/5 column (0.5 x 5 cm [Pharmacia]) that had been equilibrated with the same buffer. The flow rate was adjusted to 0.5 ml per
min. The enzyme was eluted with 30 ml of a linear gradient of 0 to
500 mM NaCl in 30 mM Tris-HCl, pH 7.4, and 0.5-ml fractions
were collected. The enzyme preparation in fractions containing sucrose synthase activity (eluting at ~200 mM NaCl) migrated as a
single band in SDS-PAGE (see Fig. 2A). One unit of sucrose synthase activity was defined as one /imol of sucrose formed per min
at 30°C.
Assays—Sucrose synthase activity in the synthesis direction
was determined using invertase digestion and the mutarotase-glucose oxidase method (glucose CH-test Wako kit, Wako-Chemicals). The reaction mixture contained 50 mM fructose, 10 mM
UDP-glucose, enzyme protein, 18 mM Tris-HCl, pH 7.5, 0.6 mM
DTT, and 0.6 mM EDTA in a total volume of 50 /il. After incubation at 30°C for 5 min, the reaction was stopped by boiling. Then
the sucrose formed was degraded by 2 units of invertase (WakoChemicals) at 37°C for 5 min, and oxidized glucose was monitored at 505 nm. One unit of invertase activity was defined as one
ftmo\ of sucrose hydrolyzed per min at 20°C. For the assay of
UDP-glucose formation, the reaction mixture contained 10 mM
UDP, 100 mM sucrose, 10 mM Tris-HCl, pH 7.5, and the sample
to be assayed in a total volume of 20^1. The reaction mixture was
incubated at 30°C for 30 min, and after the reaction was stopped
by boiling, UDP-glucose was determined essentially according to
the procedure of Tochikura et al. (1968). The reaction mixture contained 0.15 mM NAD, 65 mM glycine/NaOH buffer, pH 8.6,
0.005 units of UDP-glucose dehydrogenase (Sigma), and the sample to be assayed in a total volume of 0.3 ml. The reaction mixture
was incubated at 25°C for 30 min and measured by absorbance at
340 nm. This cleavage activity was also assayed by measuring fructose produced from sucrose in the reaction mixture as described
by Somogyi (1952). The apparent Km values for sucrose, UDP-glucose and fructose were measured as described previously at pH 7.5
with 20 to 100 mM, 0.2 to 1 mM, and 2 to 10 mM substrate solution, respectively (Nakai et al. 1997). The reaction products were
separated with paper electrophoresis, and analyzed by autoradiography or bio-imaging on a Fujix Bas 2000 (Fuji Photo Film) as described previously (Nakai et al. 1997). Protein was determined by
the method of Bradford (1976), using bovine serum albumin
(Sigma) as the standard.
The in vitro phosphorylation of the recombinant sucrose synthases from mung bean was performed with a soluble, ~55-kDa
Ca2+-dependent protein kinase (CDPK) preparation partially purified from soybean root nodules (Zhang and Chollet 1997). In a 40-
1
2
3
4
5
6
7
8
9 10 11
Amino Add Sequence : Met Ala Ttir AspArg Leu Thr Arg Val His Ser
Gene Sequence
12 13
14 15 16
Leu Arg Glu Arg Leu
-
: ATG GCT ACC GAT CGT TTG ACC CGT GTT CAC AGT CTC CGT GAG AGG CTT
Wild-type
Oligonucleotide (N-ter2):
GCT ACC GAT CGT TTG ACC CG
S e r - * Asp
Oligonucleotide
:
GCT ACC GAT CGT TTG ACC CGT GTT CAC GAT CTC CGT GAG AGG C
Asp
Ser-» Glu
Oligonucleotide
:
GCT ACC GAT CGT TTG ACC CGT GTT CAC GAA CTC CGT GAG AGG C
Glu
* • •
Fig. 1 N-Terminal amino acid sequence of mung bean sucrose synthase and synthetic oligonucleotides used for site-directed mutagenesis of Ser". The nucleotide sequences of the two oligonucleotides are shown with the asterisks indicating mismatches between the gene sequence and oligonucleotide.
Affinity for sucrose of sucrose synthase
/j\ reaction mixture, about 10 fig of purified wild-type or mutant
sucrose synthase was incubated with the appropriate amount of
nodule CDPK, 50 mM HEPES-KOH, pH 7.5, 5 mM MgCl2, and
5 fxCi [y-32P]ATP (3 Ci mmol"' from Amersham) or 2 mM ATP
for 30 min at 30°C in the presence of 1 mM Ca 2+ . The 32P reaction
mixture was immediately subjected to boiling in SDS-sample buffer and SDS-PAGE (Laemmli 1970). The dried gel was autoradiographed with X-ray film (Kodak) at room temperature.
A
(kDa)
212
170
116
Results and Discussion
Purification and properties of wild-type and mutant sucrose synthases—Three kinds of recombinant sucrose synthases were purified to electrophoretic homogeneity by
sequential gel nitration and anion-exchange FPLC (Pharmacia). The wild-type [Ser" (see also Nakai et al. 1997)],
SI ID, and SHE enzymes appeared as a homogeneous
~95-kDa polypeptide following SDS-PAGE (Fig. 2A), and
their specific activities were 0.048, 0.044, and 0.045 units
(mg protein)" 1 , respectively. The final yield of each purified enzyme was about 340, 150, and 220 fig from 8g of
E. coli cells expressing the wild-type, SI ID, and SHE enzymes, respectively. All the purified recombinant enzymes
eluted at a position calibrated as ~35O kDa on a Superose
6 HR 10/30 size-exclusion column (equilibrated with 30
mM Tris-HCl, pH 7.5, containing 100 mM NaCl, 0.1 mM
EDTA, and 0.1 mM DTT), as shown previously for the
authentic mung bean sucrose synthase (Delmer 1972).
These results indicate that the recombinant sucrose synthases have a similar subunit mass and homotetrameric
structure as the plant enzyme.
B
M 1
2 3
1 2
•»
—
76
—""""
53
—
*
Fig. 2 SDS-PAGE analysis of the three recombinant mung bean
sucrose synthases. (A) Coomassie blue-stained gel. (B) Autoradiograph of the three recombinant enzymes initially phosphorylated
with soybean nodule CDPK and Mg-[y-32P]ATP, and then resolved by SDS-PAGE. Lane 1, wild-type (Ser11); lane 2, SI ID;
lane 3, S11E;M, molecular mass markers. Protein loaded was
about 0.5 /ig in each well.
bean enzyme with Mg-[y-32P]ATP and the partially purified
legume nodule protein kinase resulted in the incorporation
of 32P into the ~95-kDa subunit (Fig. 2B, lane 1). In contrast, neither the SHD nor SI IE mutant enzyme was phosphorylated in vitro by this CDPK (Fig. 2B, lanes 2 and 3).
Thus, Ser" of mung bean sucrose synthase is the only phosphorylatable residue in this target enzyme, as reported previously for its homolog in the maize SS-2 isoform of sucrose synthase, i.e., Ser15 (Huber et al. 1996). Notably, like
the mung bean enzyme (Fig. 1; Arai et al. 1992), the root
nodule isoforms of sucrose synthase in Vicia faba L. and
soybean also share an identical TV-terminal phosphorylation motif of -Arg-Val-His-Ser"-Leu- (Kuster et al. 1993,
Zhang et al. 1997).
Phosphorylation of recombinant enzymes by legume
nodule CDPK—It has previously been reported that plant
sucrose synthase in two physiologically distinct "sink" tissues [developing maize leaves (Huber et al. 1996) and soybean root nodules (Zhang and Chollet 1997)] is phosphorylated on a target Ser residue by a ~ 5 5 - to 65-kDa CDPK.
Indeed, incubation of the purified recombinant Ser" mung
Table 1 Kinetic properties of recombinant sucrose synthases
Sucrose synthase
UDP-glucose
Wild-type (Ser")
Km (mM)
Fructose
Sucrose
0.40*
7.8*
161*
0.36
4.6
61
S11D
0.91
5.9
56
SHE
0.45
Wild-type (Ser"-P)
Seedlings
a
0.21
3
23
3.2
c
2.0
c
17d
" Phosphorylated in vitro by the soybean nodule CDPK and Mg-ATP in the presence of 1 mM Ca24
for 30 min at 30°C.
* Data from Nakai et al. (1997).
c
Data from Grimes et al. (1970).
d
Data from Delmer (1972).
1340
Affinity for sucrose of sucrose synthase
Table 2 Kinetic properties for sucrose of recombinant sucrose synthases
at'"^m
Sucrose synthase
Table 3 Effect of phenolic glycosides on sucrose synthesis with SHE mutant enzyme
Relative activity (%)"
Wild-type (Ser")
SHE
Inhibitor"
Wild-type (Ser")
0.85
5.3
None (control)
Wild-type (Ser"-P)
0.39
6.4
SI ID
0.27
4.8
SHE
0.38
16.5
Kinetic analysis—As shown in Table 1, the apparent
Km value for sucrose of the wild-type (Ser") sucrose synthase was decreased by a factor of 2.6 following its in vitro
phosphorylation by the nodule CDPK. The value was also
decreased by factors of 2.9 and 7.0 in the SI ID and SHE
mutant enzymes, respectively. In contrast, the apparent Km
values for UDP-glucose and fructose were similar in the
wild-type (Ser"), phosphorylated wild-type (Ser"-P), and
two mutant enzymes (Nakai et al. 1997). It should be noted
that the affinity for sucrose of the SI ID enzyme was similar
to that of the phosphorylated enzyme. However, the fccat
value with sucrose was decreased in the Ser"-P and mutant
enzymes relative to the wild-type (Ser") enzyme (Table 2).
The kcat/Km value of the phosphorylated wild-type enzyme
(Ser"-P) was about 20% higher than that of the wildtype enzyme. These findings confirm the previous observations (Huber et al. 1996, Winter et al. 1997) that the phosphorylation (or anionic) state of a specific TV-terminal Ser
residue at position 11 (mung bean sucrose synthase) or 15
(maize sucrose synthase) affects the enzyme's apparent affinity for sucrose. As shown in Table 1, replacement of Ser"
with Glu" increased the affinity for sucrose to produce
UDP-glucose. The kcat/Km ratio, which reflects the catalytic
100
100
Arbutin
37
20
Phenyl-yS-glucoside
63
52
° Assayed by using 25 mM fructose and 5 mM UDP-glucose at
30°C for 5 min in the absence (control) or presence of arbutin
(5.5 mM) or phenyl-/?-glucoside (1 mM).
* Relative activity was estimated as the amount of sucrose present
in the reaction mixture.
efficiency of the SHE mutant enzyme with sucrose, exceeded that of the wild-type enzyme by a factor of 3.1 and was
superior to the phosphorylated and SI ID enzymes by factors of 2.6 and 3.4, respectively (Table 2). These findings
show that the SHE mutant enzyme is superior to both the
phosphorylated sucrose synthase and the related SI ID mutant enzyme.
Enzymatic properties of the SHE mutant enzyme—
Since the SI IE mutant enzyme showed the lowest Km value
and highest kCM/Km ratio with sucrose of the four enzyme
forms examined (Table 1, 2), the enzymatic properties of
the SI IE enzyme were further characterized. The initial velocity of the SHE mutant enzyme for UDP-glucose formation from sucrose was higher than that of the wild-type
(Ser") enzyme (Fig. 3A), although the two recombinant
enzymes had a similar velocity for sucrose formation
(Fig. 3B). This is in agreement with the significant activation of the sucrose cleavage reaction by replacement of
Ser" with Glu" (Table 2). The activation may also cause
the different levels of UDP-glucose formation in the reaction mixture in equilibrium. These findings show that the
SHE mutant enzyme has been specifically activated for
UDP-glucose formation from sucrose by site-directed mutaTable 4 Nucleotide specificity of SHE mutant enzyme
Nucleotide'
50
100
150
Time (min)
200
250
10
20
30
40
Time (min)
Fig. 3 Time courses of UDP-glucose and sucrose formation. Incorporation (nmol) is expressed as the conversion of substrates
into products during sucrose cleavage (panel A) and synthesis
(panel B) at 30° C. An equal amount of purified protein was used
in each reaction mixture. The plotted data points show the wildtype (Ser") (o) and SI IE (•) enzymes. A: UDP-glucose formation
from 100 mM [14C]sucrose and 10 mM UDP; B: sucrose formation from 10 mM UDP-[l4C]glucose and 50 mM fructose.
SHE
Relative activity {%) *
Wild-type (Ser")
UDP
100
100
ADP
43
56
CDP
28
22
GDP
TDP
9.9
37
1.9
20
" Assayed using 250 mM sucrose and 10 mM UDP, ADP, CDP,
GDP, or TDP at 30°C for 30 min.
6
Relative activity was estimated as the amount of fructose
liberated in the reaction mixture.
Affinity for sucrose of sucrose synthase
genesis.
Sucrose formation by the SI IE mutant enzyme was determined in the presence of arbutin or phenyl-yS-glucoside
since these two phenolic glycosides are inhibitory to sucrose synthase activity for sucrose formation (Slabnik et al.
1968). The activity of the wild-type enzyme was inhibited in
the presence of 5.5 mM arbutin or 1 mM phenyl-/?-glucoside by 50 to 80%, whereas the level of activity of the SI IE
enzyme was somewhat more resistant to these phenolic glycosides (Table 3).
The effect of various nucleoside diphosphates on
the cleavage reaction was determined by monitoring the
amount of fructose formed. Several nucleoside diphosphates were effectively used in the order UDP > ADP >
T D P > C D P > G D P by both recombinant enzymes (Table
4). This is in agreement with an earlier observation (Delmer
1972) that the affinity for ADP was higher in the authentic
enzyme from mung bean seedlings than that for TDP, although TDP is a more favorable substrate than ADP for a
sucrose synthase from rice grains (Elling et al. 1993) and
sugar beet roots (Milner and Avigad 1965). However, the
SHE mutant enzyme uses TDP at about the same level as
ADP. This shows that replacement of Ser11 with Glu"
alters the enzymes nucleotide specificity to a modest extent.
In summary, modification of the //-terminal region of
wild-type plant sucrose synthase by in vitro phosphorylation of a target Ser residue at position 11 (legumes) or 15
(maize leaves) modestly affects the enzyme's apparent
Km for sucrose in the cleavage reaction [Table 1, and
(Huber et al. 1996, Winter et al. 1997)]. Because this
Km (sucrose) effect is functionally mimicked by the introduction of a monoanionic (1~) side chain at this position in the S11D/E mutant enzymes, we postulate that the
introduction of negative charge (2~ or 1"") into this domain
is perhaps somehow involved in this perturbation of the
cleavage activity of sucrose synthase. However, this hypothesis must be further investigated by the use of neutral substitutions (e.g., S11A, S11C) at position 11 in the mung bean
enzyme.
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1341
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(Received April 1, 1998; Accepted September 30, 1998)