©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Removal of Feedback Inhibition of -Pyrroline-5-carboxylate Synthetase, a Bifunctional Enzyme Catalyzing the First Two Steps of Proline Biosynthesis in Plants (*)

(Received for publication, May 30, 1995; and in revised form, June 21, 1995)

Chun-sheng Zhang (1) (2) Qin Lu (2) Desh Pal S. Verma (1) (2) (3)(§)

From the  (1)Department of Plant Biology, (2)Plant Biotechnology Center, and (3)Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Delta^1-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 [^14C]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.


INTRODUCTION

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, Delta^1-pyrroline-5-carboxylate synthetase (P5CS) (^1)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) .


MATERIALS AND METHODS

Bacterial Strain and Plasmid

E. coli strain CSH26 (ara, Delta (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 beta-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(4))(2)SO(4). 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 beta-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(2), 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(3) 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.

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 Delta^1-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(2)PO(4), 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 [^14C]glutamate. Root tissue from Vigna seedling (5 days old) was homogenized in extraction buffer (50 mM Tris at pH 7.0, 10 mM beta-mercaptoethanol, 300 mM sucrose, and 5 mM MgCl(2)). The extract was centrifuged, and supernatant was fractionated by 35% saturation of (NH(4))(2)SO(4). 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(2), 10 mM ATP, 5 mM NADPH, 0.1 µCi of [^14C]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, [^14C]glutamate, and [^14C]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(r) 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.


RESULTS

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.




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 beta-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(4))(2)SO(4) 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 Kand V(max) 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.



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 [^14C]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).


Figure 5: [^14C]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, [^14C]proline; lane2, [^14C]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.



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.


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) .






DISCUSSION

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(4))(2)SO(4) 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(4))(2)SO(4) 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 [^14C]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.


FOOTNOTES

*
This study was supported by United States Department of Agriculture Grants 90-37280-5596 and 92-37100-7648 (to D. P. S. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Room 240, Rightmire Hall, Plant Biotechnology Center, Ohio State University, 1060 Carmack Rd., Columbus, OH 43210. Tel.: 614-292-3625; Fax: 614-292-5379; dverma{at}magnus.acs.ohio-state.edu.

(^1)
The abbreviations used are: P5CS, Delta^1-pyrroline-5-carboxylate synthetase; -GK, -glutamyl kinase; GSA, glutamic acid-5-semialdehyde; P5C, Delta^1-pyrroline-5-carboxylate; P5CR, Delta^1-pyrroline-5-carboxylate reductase; TLC, thin layer chromatography; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

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.


REFERENCES

  1. Adams, E., and Frank, L. (1980) Annu. Rev. Biochem. 49,1005-1061 [CrossRef][Medline] [Order article via Infotrieve]
  2. Hanson, A. D., and Hitz, W. D. (1982) Annu. Rev. Plant Physiol. 33,163-203
  3. Delauney, A. J., and Verma, D. P. S. (1993) Plant J. 4,215-223 [CrossRef]
  4. Delauney, A. J., Hu, C. A., Kishor, P. B. K., and Verma, D. P. S. (1993) J. Biol. Chem. 268,18673-18678 [Abstract/Free Full Text]
  5. Smith C. J., Deutch, A. H., and Rushlow, K. E. (1984) J. Bacteriol. 157,545-551 [Medline] [Order article via Infotrieve]
  6. Hu, C. A., Delauney, A. J., and Verma, D. P. S. (1992) Proc. Natl. Acad. Sci. 89,9354-9358 [Abstract]
  7. Delauney, A. J., and Verma, D. P. S. (1990) Mol. Gen. Genet. 221,299-305 [Medline] [Order article via Infotrieve]
  8. Szoke, A., Miao, G. H., Hong, Z., and Verma, D. P. S. (1992) Plant Physiol. 99,1642-1649
  9. Csonka, L. N. (1981) Mol. Gen. Genet. 182,82-86 [Medline] [Order article via Infotrieve]
  10. Csonka, L. N., Gelvin, S. B., Goodner, B. W., Orser, C. S., Siemieniak, D., and Slightom, J. L. (1988) Gene (Amst.) 64,199-205 [CrossRef][Medline] [Order article via Infotrieve]
  11. Dandekar, A. M., and Uratsu, S. L. (1988) J. Bacteriol. 170,5943-5945 [Medline] [Order article via Infotrieve]
  12. Rudulier, D. L., Strom, A. R., Dandeker, A. M., Smith, L. T., and Valentine, R. C. (1984) Science 224,1064-1068
  13. Hayzer, D. J., and Leisinger, T. (1980) J. Gen. Microbiol. 118,287-293 [Medline] [Order article via Infotrieve]
  14. Mezl, V. A., and Knox, W. E. (1976) Anal. Biochem. 74,441-447 [Medline] [Order article via Infotrieve]
  15. Laurent, T. C., and Killander, J. (1964) J. Chromatogr. 14,317-330 [CrossRef]
  16. Harlow, E., and Lane, D. (1988) in Antibodies: A Laboratory Manual , pp. 61-70, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  17. Higchi, R., Krummel, B., and Saiki, R. K. (1988) Nucleic Acids Research 16,7351-7367 [Abstract]
  18. Baich, A. (1969) Biochem. Biophys. Acta 192,462-467 [Medline] [Order article via Infotrieve]
  19. Hayzer, D. J., and Leisinger T (1981) Biochem. J. 197,269-274 [Medline] [Order article via Infotrieve]
  20. Gamper, H., and Moses, V. (1974) Biochim. Biophys. Acta 354,75-87 [Medline] [Order article via Infotrieve]
  21. Yoshia, Y., Kiyosne, T., Katagiri, T., Ueda, H., Mizoguchi, T., Yamaguchi-Shinozaki, K., Wada, K., Harada, Y., and Shinozaki, K. (1995) Plant J. 7,751-760 [CrossRef][Medline] [Order article via Infotrieve]
  22. Li, W., and Brandniss, M. C. (1992) J. Bacteriol. 174,4148-4156 [Abstract]
  23. Kishor, P. B. K., Hong, Z., Miao, G-H., Hu, A. C-A., and Verma, D. P. S. (1995) Plant Physiol. 108,1387-1394 [Abstract/Free Full Text]
  24. Stewart, C. R., Boggess, S. F., Aspinall, D., and Paleg, L. G. (1977) Plant Physiol. 59,930-932
  25. Rayapati, P. J., and Stewart, C. R (1991) Plant Physiol. 95,787-791
  26. Boggess, S. F., Stewart, C. R., Aspinall, D., and Paleg, L. (1976) Plant Physiol. 58,398-401
  27. Boggess, S. F., Aspinall, D., and Paleg, L. (1976) Aust. J. Plant Physiol. 3,513-525

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