(Received for publication, May 30, 1995; and in revised form, June 21, 1995)
From the
-Pyrroline-5-carboxylate synthetase (P5CS)
catalyzes the first two steps in proline biosynthesis in plants. The Vigna aconitifolia P5CS cDNA was expressed in Escherichia
coli, and the enzyme was purified to homogeneity. The Vigna P5CS exhibited two activities,
-glutamyl kinase (
-GK)
and glutamic acid-5-semialdehyde (GSA) dehydrogenase. The
-GK
activity of the P5CS was detected by the hydroxamate assay and by a
[
C]glutamate assay. The native molecular mass of
the P5CS was 450 kDa with six identical subunits. The Vigna P5CS showed a K
of 3.6 mM for glutamate, while the K
for ATP
was 2.7 mM. The
-GK activity of the P5CS was
competitively inhibited by proline, while its GSA dehydrogenase
activity was insensitive to proline. In addition, a protein inhibitor
of the P5CS was observed in the plant cell. Western blot showed that
the level of the P5CS was enhanced in Vigna root under salt
stress. A single substitution of an alanine for a phenylalanine at
amino acid residue 129 of the P5CS resulted in a significant reduction
of proline feedback inhibition. The 50% inhibition values of
-GK
activity of the wild type and the mutant P5CS were observed at 5 mM and 960 mM proline, respectively. The other properties of
the mutant P5CS remained unchanged. These results may allow genetic
manipulation of proline biosynthesis and overproduction of proline in
plants for conferring water stress tolerance.
Proline is accumulated in plants under drought and salinity
stress in a number of species and is thought to play an important role
in plant cells for adaptation to water
stress(1, 2, 3) . In plants, proline is
synthesized from either glutamate or ornithine(1, 4) .
We have demonstrated that the glutamate pathway is predominant under
the condition of osmotic stress(4) . In Vigna
aconitifolia, the first two steps of the proline biosynthesis from
glutamate are catalyzed by a single bifunctional enzyme,
-pyrroline-5-carboxylate synthetase (P5CS) (
)with apparent activities of
-glutamyl kinase
(
-GK) and glutamic acid-5-semialdehyde (GSA) dehydrogenase (or
-glutamyl phosphate reductase). Two separate enzymes,
-GK and
GSA dehydrogenase, are involved in the production of GSA in Escherichia coli. In E. coli, purified
-GK
showed no detectable activity, and the addition of the GSA
dehydrogenase revealed the
-GK activity(5) . The product
(
-glutamyl phosphate) of the first enzyme was suggested to remain
in the enzyme-bound state and was rapidly converted to GSA by GSA
dehydrogenase, which forms a complex with the
-GK. The GSA
produced by these reactions is spontaneously converted into
pyrroline-5-carboxylate (P5C), which is then reduced by P5C reductase
(P5CR) to proline. The cDNAs encoding P5CS and P5CR have been isolated
from plants(6, 7) . Expression of the P5CR cDNA in
transgenic tobacco resulted in a 200-fold increase in the P5CR
activity, but the proline level in transgenic plants was not
significantly altered(8) . This result indicated that P5CR is
not the rate-limiting enzyme in proline biosynthesis in plants. The
-GK activity of the Vigna P5CS was sensitive to proline
inhibition, indicating that the P5CS may be the rate-limiting step in
proline pathway in plants(6) .
It has been demonstrated that
proline biosynthesis in bacteria is regulated by the end product
inhibition of the -GK activity(5) . A Salmonella
typhimurium mutant resistant to the toxic proline analog, L-azetidine-2-carboxylic acid, accumulated proline and showed
enhanced tolerance to osmotic stress (9) . The mutation was due
to a change of an aspartate (at position 107) to asparagine in the
-GK, resulting in a mutant
-GK, which was much less sensitive
to proline inhibition(10, 11) .
Alignment of the
protein sequences between the Vigna P5CS and the E. coli -GK and GSA dehydrogenase showed ( Fig. S1and (6) ) that the two enzymatic domains overlap in the Vigna P5CS protein, and the putative amino acid residue implicated in
the feedback inhibition of the E. coli
-GK enzyme at
position 107 (boldface) was found to be conserved in Vigna P5CS (at position 128; boldface).
Figure S1: Scheme I.
We reasoned that
site-directed mutagenesis of the corresponding feedback inhibition
region of -GK domain in the Vigna P5CS may yield alleles
able to retain high levels of the enzyme activity as the concentration
of the end product of the pathway, proline, increases. We found that
the conserved aspartate residue (at position 128) in the Vigna P5CS is not involved in the feedback inhibition, and we identified
two other residues that interfere with the proline binding. One of
these practically eliminated the feedback inhibition by proline.
The
P5CS enzyme has not been characterized in plants or animals, and the
studies on proline biosynthesis have been limited. This paper describes
the purification, kinetic studies, and mutagenesis of the Vigna P5CS. It was demonstrated that the -GK activity of the P5CS
is subject to feedback inhibition by proline and ADP, respectively. The
level of the P5CS was increased in Vigna roots treated with
NaCl. Removal of the feedback inhibition of P5CS followed by
overproduction of such a mutant enzyme in transgenic plants is expected
to cause high level accumulation of proline. The latter may render
plants capable of withstanding osmotic stress imposed by drought or
salinity as proline acts as an osmoprotectant(12) .
The GSA dehydrogenase activity of Vigna P5CS was assayed as
described by Hayzer and Leisinger(13) . The GSA dehydrogenase
activity was not detectable in the forward (biosynthetic) direction
because of the lability of -glutamyl phosphate(13) . We
measured the reverse reaction by phosphate-dependent reduction of
NADP
with glutamic acid-5-semialdehyde (derived from
equilibrium with
-pyrroline-5-carboxylate) as the
substrate. The reaction mixture contained the following in a final
volume of 0.3 ml at pH 7.0: 2.5 mM P5C prepared as described
earlier(8) , 1 mM NADP
, 100 mM KH
PO
, 50 mM imidazole base, and
the enzyme plus water. The increase in the absorbance at 340 nm was
recorded at room temperature against a blank identical to the above but
lacking inorganic phosphate. The concentration of P5C was determined
with o-aminobenzaldehyde as described by Mezl and
Knox(14) .
We developed a more sensitive assay for the
-GK activity of the P5CS using
[
C]glutamate. Root tissue from Vigna seedling (5 days old) was homogenized in extraction buffer (50
mM Tris at pH 7.0, 10 mM
-mercaptoethanol, 300
mM sucrose, and 5 mM MgCl
). The extract
was centrifuged, and supernatant was fractionated by 35% saturation of
(NH
)
SO
. The pellet was dissolved in
the extraction buffer, dialyzed against the same buffer, and assayed.
The reaction contained the following in a final volume of 20 µl at
pH 7.0: 50 mM Tris, 20 mM MgCl
, 10
mM ATP, 5 mM NADPH, 0.1 µCi of
[
C]glutamate (DuPont NEN), and enzyme samples
plus water. The reaction mixture was incubated at 37 °C for 10 min
and then chilled on ice. An aliquot (2 µl) of the reaction mixture
was resolved by thin layer chromatography (TLC) on a Silica gel
(Analtech, Inc.). P5C, glutamine, [
C]glutamate,
and [
C]proline (DuPont NEN) were used as
standards. The P5CR enzyme was purified from a proline mutant of E.
coli carrying soybean P5CR cDNA(8) . The Silica gel was
developed with a mobile phase (phenol:water:acetic acid, 75:25:5,
w/v/v) containing 0.3% (w/v) ninhydrin in a saturated chamber. After
development the gel was dried at 65 °C for 20 min, wrapped with
Saran Wrap and analyzed on a PhosphorImager (Molecular Dynamics) or
exposed to x-ray film.
Figure 1:
A, DE-52 anion exchange
chromatography of Vigna P5CS expressed in E. coli.
The combined fraction from Sephadex G-50 was applied to a DE-52 column,
and proteins were eluted by 50-300 mM NaCl linear
gradient. B, hydroxylapatite chromatography of Vigna P5CS expressed in E. coli. The combined fraction
(28-40) from DE-52 column was applied to a hydroxylapatite
column, and the proteins were eluted stepwise with 90 (fractions
9-17) and 180 (fractions 18-27) mM potassium
phosphate, pH 7.2, containing 2 mM -mercaptoethanol. The
P5CS was present in the fractions with 180 mM potassium
phosphate. Protein concentration was monitored at 280 nm. Activity, nmol of
-glutamyl hydroxamate formed per
min.
Figure 2:
SDS-PAGE showing the purification of Vigna P5CS. Lane1, protein markers in kDa; lane2, crude extract (25 µg) of E. coli strain CSH26; lane3, crude extract (25 µg)
of E. coli strain CSH26 carrying pVAB2; lane4, proteins (12 µg) from 30% saturation of
(NH)
SO
precipitation; lane5, concentrated fraction (5 µg) from DE-52 column; lane6, active fraction (2 µg) from
hydroxylapatite column.
Figure 3: The GSA dehydrogenase activity of Vigna P5CS and its sensitivity to proline inhibition. 3.0 µg of the purified P5CS was used in each assay. The enzyme activity was measured as described under ``Materials and Methods.'' Note that the GSA dehydrogenase activity of the P5CS was not affected in the presence of 100 mM proline.
Plots of -GK activity of P5CS versus glutamate concentration displayed typical Michaelis-Menten
kinetics. Double-reciprocal plots were used to estimate the K
and V
values for
glutamate, and the values obtained were 3.6 mM and 13.3
µmol of
-glutamyl hydroxamate min
mg
. Plots of the
-GK activity versus ATP concentration also displayed typical Michaelis-Menten
kinetics, and the K
for ATP was found to
be 2.7 mM. The
-GK activity of the P5CS was sensitive to
proline and its analog, 3,4-dehydroproline. A 50% inhibition (in the
presence of 50 mM glutamate) of the
-GK was observed in
the presence of 5.0 mM proline or 4.5 mM 3,4-dehydroproline. Enzyme kinetics of the
-GK at different
proline or 3,4-dehydroproline concentrations indicated that both are
competitive inhibitors, and the estimated K
for proline was 1.0 mM (Fig. 4A).
In addition, the
-GK activity of the P5CS was also inhibited by
ADP, whereas AMP and GMP had no effect (data not shown). ADP was found
to be a mixed competitive inhibitor, and the estimated K
for ADP was 6.4 mM (Fig. 4B).
Figure 4:
The effects of proline and ADP on the
-GK activity of Vigna P5CS. 3.0 µg of the purified
P5CS was used in each assay. A, double-reciprocal plots of
-GK activity of purified Vigna P5CS versus glutamate at different concentrations of proline. The result
showed that the
-GK activity was competitively inhibited by
proline. B, double-reciprocal plots of
-GK activity of
purified Vigna P5CS versus ATP at different
concentrations of ADP. Note the mixed competitive inhibition of the
-GK activity by ADP. Activity, nmol of
-glutamyl
hydroxamate formed per min.
Figure 5:
[C]Glutamate assay
for P5CS activity. The assay was conducted as described under
``Materials and Methods.'' The positions of the standard
amino acids and TLC start and front are indicated on the leftside. Lane1,
[
C]proline; lane2,
[
C]glutamate; lane3, the root
extract of Vigna (25 µg); lane4, Vigna root extract (25 µg) plus purified P5CS (0.1
µg); lane5, purified P5CS (0.1 µg); lane6, the boiled root extract of Vigna (25 µg)
plus purified P5CS (0.1 µg); lane7, purified
P5CS (0.1 µg) plus purified P5CR (0.4 µg); lane8, purified P5CS (0.1 µg) without ATP in the reaction
mixture.
Polyclonal antibodies raised against the purified P5CS were used to detect the native P5CS in Vigna roots. The P5CS antibody reacted with a protein band from the extract of stressed roots, the intensity of which was much higher than that from the extract of unstressed roots (Fig. 6), indicating that the amount of the P5CS in the root was enhanced by salt stress. The difference between the subunit sizes of expressed P5CS and the native Vigna root P5CS is apparently due to the addition of amino acid residues at the N terminus of the expressed enzyme from the expression vector in which the P5CS cDNA was fused with the lac Z promoter (see ``Discussion'').
Figure 6: The effect of NaCl on the level of P5CS in Vigna roots. The Western blot was performed as described under ``Materials and Methods.'' Lane1, crude extract (1 µg) of E. coli strain CSH26; lane2, crude extract (1 µg) of E. coli strain CSH26 carrying pVAB2; lane3, purified Vigna P5CS (0.1 µg); lane4, the root extract (65 µg) of Vigna treated with 200 mM NaCl; lane5, the root extract (65 µg) of Vigna without salt stress.
Figure 7:
Amino acid
substitutions and their effect on the feedback inhibition of Vigna P5CS by proline. A, the numbers on the top correspond to the nucleotide sequence (aligned by the asterisks, see (6) ). The numbers on the bottom correspond to the amino acid positions in the P5CS
protein. All six amino acids were replaced by an alanine individually.
The underlined amino acids represent the single substitution
in the mutant alleles that reduced proline inhibition. The substitution
of the aspartate at position 128, the putative residue involved in
proline interaction ( Fig. S1and (6) ), had no effect on
the feedback inhibition of the enzyme activity. B, the effect
of proline on the activities of purified E. coli -GK
(
), the P5CS (
), and the P5CSF129A (
). A hydroxamate
assay containing 50 mM glutamate was conducted in the presence
of different concentrations of proline. The curve of E.
coli
-GK was replotted from the data in (5) .
We described a purification procedure for a bifunctional
enzyme, P5CS, catalyzing the first two steps in proline biosynthesis in
plants (6, 21) . The purified E. coli -GK showed no detectable activity using the hydroxamate
assay, but the production of
-glutamyl hydroxamate could be
restored by the addition of purified E. coli GSA
dehydrogenase(18, 19) . It has been suggested that E. coli
-GK and GSA dehydrogenase form a complex to
afford protection to the labile
-glutamyl phosphate and to
directly transfer the intermediate from one enzyme to the other,
avoiding equilibration with the surrounding
medium(18, 20) . Such a complex has not been detected
in E. coli(5) . The Vigna P5CS is a fused
protein with two separate catalytic domains. The
-GK activity of
the purified P5CS can be detected using the hydroxamate assay. These
results supported the idea that the labile
-glutamyl phosphate
exists in an enzyme-bound state (18) and that GSA dehydrogenase
domain interacts with
-GK, effecting the release of
-glutamyl
phosphate, which can be measured as the hydroxamate derivative.
Due
to the addition of extra amino acids from the expression vector, the
molecular mass of Vigna P5CS subunit was 77 kDa as measured by
SDS-PAGE. This value was slightly higher than the molecular mass (73
kDa) deduced from the DNA sequence of the P5CS(6) . The subunit
size of native P5CS in Vigna detected by Western blot was
smaller (2 kDa) than that of the P5CS expressed in E.
coli. (Fig. 6). Therefore the molecular mass of the Vigna P5CS subunit is likely to be 75 kDa. The native
molecular mass of the P5CS is about 450 kDa as determined by gel
filtration. These results suggest that Vigna P5CS is a hexamer
of six identical subunits. Both
-GK and GSA dehydrogenase of E. coli are also hexamers with six identical
subunits(5) .
The characteristics of Vigna P5CS and
-GK of E. coli were compared in Table 2. In E.
coli, plots of the
-GK activity versus glutamate
concentration were nonhyperbolic, and the glutamate concentration
yielding half-maximal activity was 37 mM(5) . This
value is about 10-fold greater than the similar value of Vigna P5CS, suggesting that plant P5CS has higher affinity for glutamate
than E. coli
-GK.
The -GK activity of Vigna P5CS was inhibited by proline and ADP, but its GSA dehydrogenase
activity was not affected, suggesting that the
-GK is the
rate-limiting step in proline biosynthesis in plants. A similar
situation was also observed in E. coli
-GK but not in
yeast
-GK. The latter is regulated by a general amino acid control
system(22) . Proline decreases the affinity of Vigna P5CS enzyme for glutamate, but the inhibition could be partially
overcome at higher concentrations of glutamate. ADP, on the other hand,
showed a mixed competitive inhibition of
-GK activity of the P5CS,
and it is likely that ADP binds to the same site involved in ATP
binding.
The P5CS activity in Vigna roots was not
detectable. The fractionation of the root extract with
(NH)
SO
was necessary to separate
the P5CS from the glutamine synthetase activity, which is much higher
than the P5CS activity in Vigna roots. Glutamine synthetase
interferes with the P5CS assay. A 35% saturation of
(NH
)
SO
precipitated the P5CS, while
glutamine synthetase remained in solution(23) . The activity of
the purified P5CS was inhibited in the presence of the root extract,
which was eliminated by boiling the extract, suggesting the presence of
an inhibitor in the plant (Fig. 5, lanes4, 5, and 6). This may be one of the reasons why the
P5CS activity has not been detected in plants so far. The method
described here using [
C]glutamate as the
substrate is at least 50-fold more sensitive than the method of
hydroxamate assay for the P5CS activity.
We have previously shown that the expression of the P5CS mRNA in Vigna roots was enhanced by treatment of the plant with 200 mM NaCl(6) . Compared with unstressed roots, the amount of the P5CS protein in salt-treated roots was found to be enhanced. Proline biosynthesis in plants is thus primarily regulated at the transcriptional level and at the level of enzyme activity. It was also reported that proline degradation is reduced in plants under water stress(24) , and the activity of proline dehydrogenase was inhibited by KCl(25) . Therefore, it is possible that the proline accumulation in plants under stress occurs due to the increase in the amount of the P5CS and the decrease of the activity of proline dehydrogenase.
The -GK activity of the P5CS is regulated
kinetically in three ways. First, the enzyme activity responds to the
change of glutamate concentration. We had observed earlier (4) that at a high nitrogen level, the ornithine pathway for
the biosynthesis of proline was prominent, while the glutamate pathway
was reduced. Under the stress conditions (salt and drought) and low
nitrogen level, the glutamate pathway for proline biosynthesis was
dominant, and plants converted more glutamate to
proline(4, 26) . The second control involves the
inhibition of the P5CS activity by ADP. Regulation at this level would
make proline biosynthesis responsive to cellular energy level. Finally,
-GK activity of the P5CS is controlled by the end product of the
pathway, proline. This point of control is by far the most important,
since this control would ensure that there is no excess proline
production. Some earlier experiments had suggested that proline
accumulation in plants under stress may involve the loss of feedback
regulation(26, 27) . In addition, we observed the
presence of an inhibitor that may regulate the activity of the P5CS
enzyme.
In the -GK of E. coli, the change of the
aspartate at amino acid residue 107 to an asparagine led to a reduction
of proline inhibition(10, 11) . The alignment of
protein sequences between Vigna P5CS and E. coli
-GK showed that the aspartate at position 128 in the P5CS
corresponds to the aspartate at the position 107 in E. coli
-GK ( Fig. S1and (6) ). This aspartate (at
position 128) in the P5CS was changed to an asparagine, but the mutant
P5CS (P5CSD128N) showed no reduction of proline inhibition. The alanine
scanning of this region resulted in two single substitution mutants of
the P5CS, P5CSD126A, and P5CSF129A, showing the reduction of proline
inhibition. The P5CSF129A exhibited a significant increase of 50%
inhibition by proline (Fig. 7B), while other properties
of this enzyme remained unchanged (Table 3). It is likely that
the glutamate and proline binding sites are not the same but may
partially overlap or be immediately adjacent to each other so that the
binding of glutamate affects the binding of proline and vice
versa. However, we have no data to exclude the possibility that
the substitution caused a change in the conformation of the enzyme so
that the enzyme lost its allosteric properties. Obviously both
residues, the aspartate at 126 and the phenylalanine at 129, are
involved in proline binding. The phenylalanine is more important with
respect to proline binding, since the reduction of proline inhibition
obtained by the P5CSD126A is only 10% of that obtained by the
P5CSF129A. X-ray structure of the P5CS and its mutants may produce
interesting results about the mechanism of proline binding.
Overexpression of the P5CS in transgenic plants has been demonstrated
to produce more proline and render plants less sensitive to osmotic
stress(23) . Reduction of feedback inhibition of the P5CS may
further increase the accumulation of proline in transgenic plants.