| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, May 30, 1995; and in revised form, June 21, 1995) From the
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,
It has been demonstrated that
proline biosynthesis in bacteria is regulated by the end product
inhibition of the Alignment of the
protein sequences between the Vigna P5CS and the E. coli
Figure S1:
Scheme I.
We reasoned that
site-directed mutagenesis of the corresponding feedback inhibition
region of 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
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 We developed a more sensitive assay for the
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
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
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
Figure 4:
The effects of proline and ADP on the
Figure 5:
[
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
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 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 ( The characteristics of Vigna P5CS and
The The P5CS activity in Vigna roots was not
detectable. The fractionation of the root extract with
(NH 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 In the
Volume 270,
Number 35,
Issue of September 01, pp. 20491-20496, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-Pyrroline-5-carboxylate Synthetase, a Bifunctional
Enzyme Catalyzing the First Two Steps of Proline Biosynthesis in Plants (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
-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) .
-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) .
-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).
-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.
-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) .
Bacterial Strain and Plasmid
E. coli strain CSH26 (ara, (lac proBA), thi), a
proline auxotroph, was obtained from Barbara Bachmann (E. coli Genetic Stock Center, Yale University). The plasmid,
pVAB2(6) , is a pcDNA II (Invitrogen, San Diego) carrying a
full-length cDNA encoding Vigna P5CS.Purification of Vigna P5CS Expressed in E.
coli
E. coli strain CSH26 carrying pVAB2 was grown for
16 h at 37 °C in LB medium containing 80 µg/ml of ampicillin.
The cells were harvested by centrifugation, washed with cold buffer A
(30 mM Tris-HCl, pH 7.2, containing 2 mM 
-mercaptoethanol), and resuspended in the same buffer. The
cells were broken by sonication and centrifuged at 35,000 g for 20 min. The supernatant was fractionated by 30% saturation of
(NH
)
SO. After centrifugation, the
pellet was resuspended in buffer A and applied to a Sephadex G-50
column (Pharmacia Biotech Inc.). The proteins were eluted with the
buffer A, and fractions containing the P5CS activity were pooled and
applied to a DE-52 column (Whatman). Proteins were eluted by
50-300 mM NaCl linear gradient in buffer A. Fractions
with the P5CS activity were combined and applied to a hydroxylapatite
column (Bio-Rad) equilibrated with 10 mM potassium phosphate
buffer, pH 7.2, containing 2 mM -mercaptoethanol. The
column was washed with the same buffer, and proteins were eluted
stepwise with 90 and 180 mM potassium phosphate in the same
buffer. The fractions with the P5CS activity were combined and dialyzed
against buffer A containing 100 mM NaCl. The purified P5CS was
stored at -80 °C.Enzyme Assay
The P5CS activity was assayed first
by hydroxamate to detect the -GK activity as described by Hayzer
and Leisinger(13) . The reaction mixture contained the
following in a final volume of 0.1 ml at pH 7.0: 50 mML-glutamate, 20 mM MgCl
, 10 mM ATP, 100 mM hydroxamate-HCl, 50 mM Tris, and the
enzyme plus water. The reaction was started by the addition of the
enzyme. After 5 min at 37 °C the reaction was terminated by the
addition of 0.2 ml of the stop buffer (2.5 g of FeCl
and
6.0 g of trichloroacetic acid in a final volume of 100 ml of 2.5 N HCl). Precipitated proteins were removed by centrifugation, and
the absorbance at 534 nm (A) was recorded
against a blank identical to the above but lacking ATP. The amount of
-glutamyl hydroxamate was determined from the A
by the comparison with a standard curve of
-glutamyl
hydroxamate (Sigma). One unit of
-GK activity was defined as the
amount of the enzyme required to produce 1 µmol of
-glutamyl
hydroxamate/min. This assay was used during all steps of purification.
-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) .-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.Molecular Mass Determination
The native molecular
mass of Vigna P5CS expressed in E. coli was estimated
by gel filtration on a Superose-6 high performance liquid
chromatography column (1 30 cm, Pharmacia). Protein standards
were run simultaneously with the purified enzyme or separately in a
second run. The samples were applied to the column equilibrated with
buffer A containing 100 mM NaCl. The protein standards
(Bio-Rad) used were thyroglobulin (670,000), bovine gamma globulin
(158,000), chicken ovalbumin (44,000), equine myoglobin (17,000), and
vitamin B-12 (1,350). The molecular mass of the enzyme was estimated in
duplicate by means of a plot of K
for the
standards against the logarithm of the molecular mass(15) . The
subunit molecular mass of the P5CS was estimated by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE).
Antibody against Vigna P5CS
The purified P5CS was
subjected to SDS-PAGE (7.5%), and the protein band was located by
Coomassie Blue staining(16) , excised, and homogenized in
liquid nitrogen. The gel powder was mixed with an equal volume of
Freund's adjuvant (Life Technologies, Inc.), and antiserum was
prepared in rabbits. Serum obtained 10 days after the third injection
was applied to a protein A column, and the IgG fractions were eluted by
100 mM glycine-HCl, pH 3.0. Following adjustment of the pH to
7.4, the purified IgG was stored at -20 °C.Protein Extraction and Western Blot
Analysis
Vigna roots (2.5 g, fresh weight, one week
old) with or without treatment of NaCl (200 mM, 72 h) were
homogenized in liquid nitrogen and resuspended in 2 ml of buffer A
containing 1 mM phenylmethylsulfonyl fluoride. The resulting
slurry was centrifuged at 80,000 g for 10 min at 4
°C. The supernatant was saved and concentrated 10-fold using a
Centricon concentrator (M
cut-off 10,000; Amicon).
The concentrated sample was subjected to SDS-PAGE (7.5%), and protein
bands were transferred to nitrocellulose membranes. The P5CS peptide
was detected by reacting with the P5CS antibody and a second antibody
using the ECL procedure (Amersham Corp.).Alanine-scanning Mutagenesis of the P5CS
The
alanine substitutions were performed using oligonucleotide-directed
mutagenesis (17) . The first two polymerase chain reactions
produced two overlapping DNA fragments, both bearing the same mutation
introduced via primer mismatch in the region of the overlap. The two
overlapping fragments were mixed and used as the template for the
second polymerase chain reaction with two flanking primers. The
fragment (700 base pairs) produced by the second polymerase chain
reaction was purified by agarose gel electrophoresis. The purified
fragment was digested with restriction enzyme HindIII and
subcloned into pVAB2 from which the corresponding wild type fragment of
the P5CS gene had been removed by HindIII digestion. The
reconstructed pVAB2 carrying the substitution was introduced into E. coli strain CSH26. Crude extracts were made from the
strains harboring the reconstructed pVAB2 and assayed for the -GK
activity of the P5CS in the presence of proline. DNA sequencing was
conducted to confirm the substitution. The P5CS enzymes carrying a
single substitution of an alanine for an aspartate at amino acid
position 126 and an alanine for a phenylalanine at amino acid position
129 were named P5CSD126A and P5CSF129A, respectively.
Purification and Properties of Vigna P5CS
The Vigna P5CS cDNA was expressed in E. coli, and the
enzyme was purified as summarized in Table 1. DEAE-cellulose
chromatography (Fig. 1A) resulted in a 27-fold
purification of the enzyme over crude extract, but this step also lost
a significant amount of the enzyme (Table 1). The enzyme was
eluted from the column around 120 mM NaCl. The hydroxylapatite
chromatography (Fig. 1B) gave a final yield of the P5CS
of 7% and represented a 54-fold purification over the crude extract (Table 1). The purified enzyme was essentially free of
contaminating proteins and appeared as a single band on SDS-PAGE (Fig. 2). The -GK activity of the purified P5CS displayed a
linear response up to 5 min with respect to the amount of protein
between 2.0 and 3.0 µg/assay. The purified enzyme lost about half
and one-third of its activity by overnight storage at 4 °C and
-20 °C, respectively, but it was stable at -80 °C
for up to 3 weeks. The maximal activity of the
-GK was obtained at
37 °C with a pH optimum between pH 6.5 and 7.5 in buffer A. The
specific activity of the GSA dehydrogenase of Vigna P5CS was
found to be 0.8 µmol of NADPH min
mg
. High concentrations of proline had no
effect on the GSA dehydrogenase activity (Fig. 3). These results
thus confirmed at enzymatic level that the Vigna P5CS is a
bifunctional enzyme with two separate enzyme activities,
-GK and
GSA dehydrogenase. The native molecular mass of Vigna P5CS was
estimated to be 450 kDa as determined by gel filtration on Superose-6
HPLC. The subunit molecular mass was estimated to be 77 kDa (see
``Discussion''), suggesting that P5CS is a hexamer with six
identical subunits.
-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.
)SO precipitation; lane5, concentrated fraction (5 µg) from DE-52 column; lane6, active fraction (2 µg) from
hydroxylapatite column.
-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).
-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.
The P5CS Level in Vigna Roots under Normal or Stress
Conditions
The P5CS activity has so far not been detected in
plants. We developed a method, using
[C]glutamate as the substrate, to detect the
P5CS activity in Vigna roots. The reaction with purified P5CS
showed the accumulation of P5C (Fig. 5, lane5), and the addition of P5CR to the reaction mixture
resulted in the production of proline (Fig. 5, lane7). No activity of the P5CS was detected in Vigna roots (Fig. 5, lane3). The root extract
added to the purified P5CS inhibited the -GK activity, but boiling
the root extract prior to the addition removed the inhibition of the
P5CS (Fig. 5, lanes4 and 6). This
suggested that there may be a protein inhibitor in the plant extract
that inhibits the P5CS activity. The
-GK activities of the P5CS
with and without the addition of the P5CR were similar (data not shown)
based on the radioactivity of P5C and proline spots resolved on TLC (Fig. 5, lanes5 and 7).
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.
Removal of Feedback Inhibition of the P5CS by
Mutagenesis
Eight substitution mutants were created and
expressed in E. coli strain CSH26. The crude extracts made
from mutants were assayed for the -GK activity in the presence of
10 mM proline. The first mutant, carrying the substitutions of
three alanines for the amino acid residues at positions 126, 127, and
128 ( Fig. S1and Fig. 7A), showed no inhibition
at 10 mM proline. The second mutant, bearing the substitutions
of three alanines for the amino acid residues at positions 129, 130,
and 131 ( Fig. S1and Fig. 7A), also showed no
inhibition of the activity in the presence of 10 mM proline.
The remaining six mutants were created by the substitution of an
alanine for the individual residues at the positions from 126 to 131,
respectively. Two of the six single substitution mutants, P5CSD126A and
P5CSF129A (Fig. 7A), showed significant reduction of
proline inhibition, while others showed no effect on proline inhibition
(data not shown). The 50% inhibition values of
-GK of the
P5CSF129A (Fig. 7B) and P5CSD126A were observed in the
presence of 960 and 85 mM proline, respectively. The K
of P5CSF129A for proline in the
presence of glutamate was 195 mM, which is about 200-fold
greater than that of the wild type P5CS (Table 3). The P5CSF129A
was also purified, and its kinetic characteristics were found to be
similar to the wild type P5CS except for the feedback inhibition.
-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) .
-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.
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) .
-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.
-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.
)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.-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.
-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.
)-pyrroline-5-carboxylate synthetase; -GK,
-glutamyl kinase; GSA, glutamic acid-5-semialdehyde; P5C,
-pyrroline-5-carboxylate; P5CR,
-pyrroline-5-carboxylate reductase; TLC, thin layer
chromatography; PAGE, polyacrylamide gel electrophoresis.
We thank Dr. Shinozaki from Riken, Japan, for
providing Arabidopsis P5CS sequence prior to its publication
and Dr. Z. Hong for help in the P5CS assay.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  L. P. Zhang, S. K. Mehta, Z. P. Liu, and Z. M. Yang Copper-Induced Proline Synthesis is Associated with Nitric Oxide Generation in Chlamydomonas reinhardtii Plant Cell Physiol., March 1, 2008; 49(3): 411 - 419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Sekine, A. Kawaguchi, Y. Hamano, and H. Takagi Desensitization of Feedback Inhibition of the Saccharomyces cerevisiae {gamma}-Glutamyl Kinase Enhances Proline Accumulation and Freezing Tolerance Appl. Envir. Microbiol., June 15, 2007; 73(12): 4011 - 4019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Deuschle, D. Funck, G. Forlani, H. Stransky, A. Biehl, D. Leister, E. van der Graaff, R. Kunze, and W. B. Frommer The Role of {Delta}1-Pyrroline-5-Carboxylate Dehydrogenase in Proline Degradation PLANT CELL, December 1, 2004; 16(12): 3413 - 3425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Terao, S. Nakamori, and H. Takagi Gene Dosage Effect of L-Proline Biosynthetic Enzymes on L-Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae Appl. Envir. Microbiol., November 1, 2003; 69(11): 6527 - 6532. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Fujita, A. Maggio, M. Garcia-Rios, C. Stauffacher, R. A. Bressan, and L. N. Csonka Identification of Regions of the Tomato gamma -Glutamyl Kinase That Are Involved in Allosteric Regulation by Proline J. Biol. Chem., April 11, 2003; 278(16): 14203 - 14210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Morita, S. Nakamori, and H. Takagi L-Proline Accumulation and Freeze Tolerance in Saccharomyces cerevisiae Are Caused by a Mutation in the PRO1 Gene Encoding {gamma}-Glutamyl Kinase Appl. Envir. Microbiol., January 1, 2003; 69(1): 212 - 219. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Sleator, C. G. M. Gahan, and C. Hill Mutations in the Listerial proB Gene Leading to Proline Overproduction: Effects on Salt Tolerance and Murine Infection Appl. Envir. Microbiol., October 1, 2001; 67(10): 4560 - 4565. [Abstract] [Full Text]
|
|
|
|
|
|
F. Hayashi, T. Ichino, M. Osanai, and K. Wada Oscillation and Regulation of Proline Content by P5CS and ProDH Gene Expressions in the Light/Dark Cycles in Arabidopsis thaliana L. Plant Cell Physiol., October 1, 2000; 41(10): 1096 - 1101. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Hong, K. Lakkineni, Z. Zhang, and D. P. S. Verma Removal of Feedback Inhibition of Delta 1-Pyrroline-5-Carboxylate Synthetase Results in Increased Proline Accumulation and Protection of Plants from Osmotic Stress Plant Physiology, April 1, 2000; 122(4): 1129 - 1136. [Abstract] [Full Text]
|
|
|
|
|
|
N. H. Roosens, R. Willem, Y. Li, I. Verbruggen, M. Biesemans, and M. Jacobs Proline Metabolism in the Wild-Type and in a Salt-Tolerant Mutant of Nicotiana plumbaginifolia Studied by 13C-Nuclear Magnetic Resonance Imaging Plant Physiology, December 1, 1999; 121(4): 1281 - 1290. [Abstract] [Full Text]
|
|
|
|
|
|
A. P. Stines, D. J. Naylor, P. B. Høj, and R. van Heeswijck Proline Accumulation in Developing Grapevine Fruit Occurs Independently of Changes in the Levels of Delta 1-Pyrroline-5-Carboxylate Synthetase mRNA or Protein Plant Physiology, July 1, 1999; 120(3): 923 - 923. [Abstract] [Full Text]
|
|
|
|
|
|
C.-a. A. Hu, W.-W. Lin, C. Obie, and D. Valle Molecular Enzymology of Mammalian Delta 1-Pyrroline-5-carboxylate Synthase. ALTERNATIVE SPLICE DONOR UTILIZATION GENERATES ISOFORMS WITH DIFFERENT SENSITIVITY TO ORNITHINE INHIBITION J. Biol. Chem., March 5, 1999; 274(10): 6754 - 6762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Fujita, A. Maggio, M. Garcia-Rios, R. A. Bressan, and L. N. Csonka Comparative Analysis of the Regulation of Expression and Structures of Two Evolutionarily Divergent Genes for Delta 1-Pyrroline-5-Carboxylate Synthetase from Tomato Plant Physiology, October 1, 1998; 118(2): 661 - 674. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
