Abstract

ATP-dependent phosphoenolpyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana: AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. AthPEPCK1 and AthPEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had no effect on AthPEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fine allosteric regulation.

Introduction

Phosphoenolpyruvate (PEP) is a key metabolic intermediate because it is a high-energy compound (ΔG = −14.8 kcal mol−1 for PEP hydrolysis) and it participates in many enzymatic reactions [1]. PEP is also important to connect a variety of metabolic pathways, including glycolysis, gluconeogenesis and organic acid metabolism [1,2]. Among the many enzymes that metabolize PEP, PEP carboxykinase (PEPCK) is particularly relevant. Based on the phosphate donor employed, PEPCKs can be classified as ATP- (EC 4.1.1.49), GTP- (EC 4.1.1.32) or PPi-dependent (EC 4.1.1.38), with different evolutionary origins [25]. The ATP-dependent PEPCK is present in bacteria, yeasts and plants. This enzyme reversibly catalyzes oxaloacetate (OAA) decarboxylation, according to the reaction: OAA + ATP ↔ PEP + ADP + CO2. PEPCK has an absolute requirement of divalent cations for its activity [6]. One metal ion acts as an essential activating cofactor, with Mn2+ the most activating [58]. A second divalent cation (either Mn2+ or Mg2+) is involved in forming the metal–nucleotide complex that constitutes the active form of the substrate [6].

In plants, PEPCK is well known for its central role in the CO2 concentrating mechanisms operating in C4 [9,10] and CAM photosynthesis [11], where it provides CO2 to the Benson–Calvin cycle by decarboxylating the OAA derived from malate or aspartate. Also, PEPCK has a crucial role during seed germination, channeling carbon released from fatty acid reserves to form sugars until the development of the photosynthetic apparatus [1216]. It has also been suggested that this enzyme is involved in nitrogen and amino acid metabolism [17,18]. Arabidopsis thaliana has two putative PEPCK genes (AT4G37870 and AT5G65690, coding for AthPEPCK1 and AthPEPCK2, respectively), which are expressed in most plant tissues [19]. The key role of PEPCK in gluconeogenesis has been clearly established in germinating seeds of this species using various experimental approaches [2023]. However, the biochemical and regulatory properties of both isozymes remain largely unknown.

In this work, we cloned the genes coding for AthPEPCK1 and AthPEPCK2 and expressed them in Escherichia coli. The recombinant enzymes thus produced were highly purified and characterized. Our results show that both enzymes are more efficient to catalyze the decarboxylation of OAA in vitro. We also identified metabolites that differentially affect the activity of AthPEPCK1 and AthPEPCK2, which could modulate both isozymes under differential physiological conditions. Our results aim to bring new insights into the biochemical regulation of PEPCKs of C3 species and to integrate these findings with the metabolic and developmental data available in the literature, to better understand the regulation of PEP metabolism in C3 plants.

Experimental

Reagents

ATP, ADP, PEP, OAA, NADH, Glc6P, l-malic acid, pyruvate kinase (PK) and malic dehydrogenase were purchased from Sigma–Aldrich. l-lactate dehydrogenase was purchased from Roche. All other reagents were of the highest available quality.

Cloning, expression and purification of AthPEPCKs

Sequences coding for AthPEPCK1 (AT4G37870) and AthPEPCK2 (AT5G65690) were retrieved from the TAIR database [24]. The gene coding for AthPEPCK1 was amplified from the pCMV-Sport6/PEPCK vector (kindly provided by Dr. C. Lacomme from INRA, CNRGV Plant Genomic Center, Toulouse, France). The sequence coding for AthPEPCK2 was de novo synthesized using optimized codons for expression in E. coli (Bio Basic). Both genes were cloned between SacI and SalI sites within the multiple cloning site 1 of the pRSFDuet-1 vector (Novagen), in frame with an N-terminal His6-tag.

AthPEPCK1 was expressed in E. coli SHuffle T7 (New England BioLabs), while AthPEPCK2 was expressed in E. coli BL21 (DE3) (Invitrogen). Cells were grown in LB medium supplemented with 50 µg ml−1 kanamycin (and 100 µg ml−1 spectinomycin in the case of SHuffle T7 cells) and protein expression was induced with 0.5 mM isopropyl-β-d-1-thiogalactopyranoside for 16 h at 25°C and 180 rpm. Cells were harvested by centrifugation, resuspended in lysis buffer [25 mM Tris–HCl pH 8.0, 300 mM NaCl, 5% (v/v) glycerol and 10 mM imidazole], disrupted by sonication and centrifuged twice at 12 000×g for 20 min at 4°C. The crude extract was loaded onto a 1-ml HisTrap HP column previously equilibrated with lysis buffer, connected to an ÄKTA Explorer 100 system (GE Healthcare). Proteins were eluted with a linear imidazole gradient (10–300 mM) in lysis buffer. Fractions containing PEPCK activity were pooled, concentrated and stored at −80°C until use. Under these conditions, both enzymes remained fully active for at least three months.

Native molecular mass determination

Protein molecular mass was determined by gel filtration chromatography using a Superdex 200 10/300 column (GE Healthcare) equilibrated with 50 mM HEPES pH 8.0 and 100 mM NaCl. A calibration curve was constructed by plotting Kav values versus log(molecular mass) of protein standards, including thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa) and ribonuclease (13.7 kDa). Kav values were calculated as (Ve − V0)/(Vt − V0), where Ve is the elution volume of the protein, V0 is the elution volume of Dextran Blue (Promega) and Vt is the total volume of the column.

Enzyme activity assays

The standard conditions for PEPCK activity measurements consisted of 100 mM HEPES–NaOH pH 7.0, 4 mM β-mercaptoethanol, 4 mM MgCl2, 10 µM MnCl2 and 0.2 mM NADH. The reaction in the direction of PEP carboxylation contained (unless otherwise specified): 90 mM KHCO3, 2 U malate dehydrogenase, 0.13 mM ADP and 10 mM PEP. The reaction in the direction of OAA decarboxylation contained (unless otherwise specified): 1 U PK, 1 U lactate dehydrogenase, 0.75 mM ATP and 0.75 mM OAA (freshly prepared). Assays were performed in 50 µl at 30°C.

Kinetic parameters were calculated using the software OriginPro 8 (OriginLab Corporation). Activity data were plotted against the concentration of the variable substrate or effector and fitted to a modified Hill equation: v = v0 + (V – v0) × CnH/(knH + CnH); where v is the initial velocity; v0 is the velocity in absence of the substrate or effector being analyzed; V is the maximal velocity (Vmax), activation or inhibition; C is the concentration of substrate or effector under study; k is the concentration of substrate or effector producing half of the maximal velocity (Km), activation (A0.5) or inhibition (I0.5); and nH is the Hill coefficient. Substrate kinetic parameters were calculated fixing the nH to 1, which turns the modified Hill equation into the classical Michaelis–Menten equation. One unit (U) is defined as the amount of enzyme that catalyzes the formation of 1 µmol of the product (PEP or OAA) per minute under the specified assay conditions (of decarboxylation or carboxylation, respectively). Allosteric effectors were assayed under standard conditions (see above). Before performing these experiments, effectors were tested on coupled enzymes to avoid unwanted effects.

Protein thermal shift assays

To analyze metabolite binding to the recombinant enzymes, we employed thermal shift assays coupled to differential scanning fluorometry according to the method described by Ericsson et al. [25]. Assays were performed in a final volume of 20 µl with 0.15 mg ml−1 protein, 4X Sypro Orange (Sigma) and 25 mM HEPES–NaOH pH 7.0 in MicroAmp fast 96-well PCR plates (Applied Biosystems). All the reactions were performed with their corresponding controls (without protein or effector). Plates were sealed with Microseal adhesive film (Bio-Rad) and heated in a StepOne Real-Time PCR System (Applied Biosystems) from 25 to 99°C, with increments of 0.4°C. Changes in fluorescence were monitored simultaneously. The wavelengths for excitation and emission were 490 and 575 nm, respectively. Melting temperature (Tm) of each sample was calculated by plotting the first derivative of the fluorescence emission (−dF/dT) as a function of temperature (T) and identifying the minimum of the curve. The shift in the melting temperature (ΔTm) was calculated subtracting each Tm to the control without effector.

Results

Arabidopsis genome contains two genes coding for putative PEPCKs [24]. The AT4G37870 gene encodes a putative protein (AthPEPCK1) of 672 amino acids with theoretical pI of 6.63 and estimated mass of 73.5 kDa, while the AT5G65690 gene codes for a putative protein (AthPEPCK2) of 671 amino acids with theoretical pI of 5.99 and estimated mass of 73.0 kDa. The sequence identity between both proteins is 78.4% (86.5% similarity), being the main differences found at the first 150 amino acids (Supplementary Figure S1). We cloned the sequences coding for AthPEPCK1 and AthPEPCK2 and the recombinant proteins were expressed with a 2 kDa tag (including the His6-tag) at the N-termini. These enzymes were purified by immobilized nickel affinity chromatography (IMAC-Ni2+), yielding highly pure (>90%) protein preparations, with molecular masses of ∼75 kDa (Figure 1A). The purified recombinant enzymes exhibited specific activity values of 3 and 5 U mg−1 (AthPEPCK1 and AthPEPCK2, respectively), when assayed in the direction of OAA decarboxylation under the standard condition described in the Experimental section. Based on their migration in the gel filtration column (Figure 1B and Supplementary Figure S2), we determined that both enzymes are hexamers of ∼440 kDa. The activity of AthPEPCKs was maximal at pH 7.0 with HEPES–NaOH in both directions of the reaction (Supplementary Figure S3), a similar behavior to that observed for other plant PEPCKs [8,2629]. We chose this buffer to perform the kinetic characterization of both enzymes because its interaction with metal ions is negligible [30] and the pH value is very close to that found in the plant cytosol (7.3 ± 0.1) [3133].

Purification and native molecular mass determination for AthPEPCK1 and AthPEPCK2.

Figure 1.
Purification and native molecular mass determination for AthPEPCK1 and AthPEPCK2.

(A) The purified AthPEPCK1 and AthPEPCK2 were analyzed by 12% SDS–PAGE; MM: molecular mass marker. (B) Superdex 200 gel filtration calibration curve constructed using the protein standards described in the Experimental section. For the samples (indicated with an arrow), the data shown are the mean of two experiments.

Figure 1.
Purification and native molecular mass determination for AthPEPCK1 and AthPEPCK2.

(A) The purified AthPEPCK1 and AthPEPCK2 were analyzed by 12% SDS–PAGE; MM: molecular mass marker. (B) Superdex 200 gel filtration calibration curve constructed using the protein standards described in the Experimental section. For the samples (indicated with an arrow), the data shown are the mean of two experiments.

PEPCK activity is dependent on both Mg2+ and Mn2+ ions; the former is necessary to form the true substrate (ATP–Mg2+) and the latter acts as an activator [8]. We analyzed the effect of increasing Mn2+ concentration at a fixed Mg2+ level, and vice versa, on AthPEPCK1 and AthPEPCK2. These experiments were done in the carboxylation direction because measuring PEPCK activity on the opposite direction requires the obligate addition of a metal ion for PK [34]. The highest activity values (∼3.5 U mg−1 for AthPEPCK1 and ∼6.0 U mg−1 for AthPEPCK2) were obtained at 1 mM Mn2+ in absence of Mg2+, whereas the presence of 0.5 mM Mg2+ decreased ∼2-fold the activity of both enzymes (Figure 2A,C). The opposite pattern was observed at low Mn2+ concentrations (0 to 0.1 mM), since the presence of 0.5 mM Mg2+ slightly increased their activity (Figure 2A,C). AthPEPCK1 and 2 scarcely responded to Mg2+ in the absence of Mn2+; however, a concentration as low as 10 μM Mn2+ produced a discrete response to Mg2+ (Figure 2B,D). Within plant cells, cytosolic Mn2+ concentration has been reported to be in the 0.3–0.8 µM range [35,36]; therefore, kinetic parameters were determined at physiological concentrations of both metal ions (4 mM Mg2+ and 10 µM Mn2+), as previously reported by Chen et al. [37].

Metal ion dependence of AthPEPCKs carboxylation activity.

Figure 2.
Metal ion dependence of AthPEPCKs carboxylation activity.

AthPEPCK1 (A) and AthPEPCK2 (C) activity was measured at increasing Mn2+ concentration in absence (squares) or presence of 0.5 mM Mg2+ (circles). AthPEPCK1 (B) and AthPEPCK2 (D) activity was measured at increasing Mg2+ concentration in the absence (up triangles) or presence of 10 μM (inverted triangles) or 100 μM of Mn2+ (diamonds). A control with 0.1 mM of EGTA was added to eliminate traces of Mn2+ (circles). Substrate concentrations were 0.3 mM ADP and 6 mM PEP. The arrows indicate the physiological concentration of metals chosen for further kinetic characterization. Data are the mean of two independent data sets ± SE.

Figure 2.
Metal ion dependence of AthPEPCKs carboxylation activity.

AthPEPCK1 (A) and AthPEPCK2 (C) activity was measured at increasing Mn2+ concentration in absence (squares) or presence of 0.5 mM Mg2+ (circles). AthPEPCK1 (B) and AthPEPCK2 (D) activity was measured at increasing Mg2+ concentration in the absence (up triangles) or presence of 10 μM (inverted triangles) or 100 μM of Mn2+ (diamonds). A control with 0.1 mM of EGTA was added to eliminate traces of Mn2+ (circles). Substrate concentrations were 0.3 mM ADP and 6 mM PEP. The arrows indicate the physiological concentration of metals chosen for further kinetic characterization. Data are the mean of two independent data sets ± SE.

Substrate kinetics for AthPEPCK1 and 2 were performed in both directions of catalysis (Table 1 and Supplementary Figures S4 and S5). Both enzymes exhibited affinity (estimated from Km values) of more than one order of magnitude higher for the binding of OAA compared with PEP. In the case of the nucleotides, ADP and ATP affinities were in the same range. In general, the specific activity and substrate affinities were higher for AthPEPCK2 than AthPEPCK1 (Table 1). Values of Vmax in the decarboxylation direction were slightly higher (up to 2-fold) compared with those observed for the carboxylation reaction. These results support that AthPEPCKs preferentially catalyze the decarboxylation of OAA than the carboxylation of PEP (see catalytic efficiencies reported in Table 1).

Table 1
Substrate kinetic parameters for AthPEPCK1 and AthPEPCK2
Enzyme Reaction direction Substrate Km (µM) Vmax (U mg−1Catalytic efficiency (× 103 M−1 s−1
AthPEPCK1 Carboxylation PEPa 3700 ± 500 1.40 ± 0.03 0.5 ± 0.1 
ADPb 79 ± 1  25 ± 2 
Decarboxylation OAAc 230 ± 20 2.6 ± 0.3 13 ± 3 
ATPd 72 ± 2  39 ± 5 
AthPEPCK2 Carboxylation PEPa 3800 ± 300 3.2 ± 0.1 1.1 ± 0.1 
ADPb 39 ± 4  100 ± 16 
Decarboxylation OAAc 100 ± 10 5.4 ± 0.1 82 ± 6 
ATPd 18 ± 3  370 ± 42 
Enzyme Reaction direction Substrate Km (µM) Vmax (U mg−1Catalytic efficiency (× 103 M−1 s−1
AthPEPCK1 Carboxylation PEPa 3700 ± 500 1.40 ± 0.03 0.5 ± 0.1 
ADPb 79 ± 1  25 ± 2 
Decarboxylation OAAc 230 ± 20 2.6 ± 0.3 13 ± 3 
ATPd 72 ± 2  39 ± 5 
AthPEPCK2 Carboxylation PEPa 3800 ± 300 3.2 ± 0.1 1.1 ± 0.1 
ADPb 39 ± 4  100 ± 16 
Decarboxylation OAAc 100 ± 10 5.4 ± 0.1 82 ± 6 
ATPd 18 ± 3  370 ± 42 

Reactions were performed as described in the Experimental section. Constants were calculated by fitting experimental data to the modified Hill equation fixing nH to 1 (Supplementary Figures S4 and S5). Reported values are the mean ± SE obtained from the fitting software. Fixed substrate concentrations used were as follows:

a

0.25 mM ADP;

b

15 mM PEP;

c

0.75 mM ATP;

d

0.75 mM OAA.

Enzymes involved in key metabolic steps are generally regulated by different mechanisms. From these, allosteric regulation plays a critical role on the fine control of enzymatic activities [3841]. To identify putative modulators of AthPEPCKs activity we performed thermal shift assays [4245], using a broad range of metabolites from several metabolic pathways that could potentially act as enzyme ligands. In our experiments, melting curves were highly reproducible (an example is shown in Supplementary Figure S6), with coefficients of variation lower than 1.5%. Among the tested metabolites, those that significantly modified the Tm of AthPEPCKs were: fructose 6-phosphate (Fru6P), glucose 6-phosphate (Glc6P), glucose 1-phosphate (Glc1P), fructose 1,6-bisphosphate (Fru1,6bisP), 3-phosphoglycerate (3PGA), inorganic pyrophosphate (PPi), the PEP-analogue N-(phosphonomethyl)glycine, pyruvate (Pyr), acetyl-CoA, citrate, malate, succinate, fumarate, l-glutamate (l-Glu), shikimate, l-arginine (l-Arg) and l-aspartate (l-Asp). Interestingly, the Tm increase for some metabolites was dependent on their concentration (Figure 3).

High-throughput screening of effectors binding to AthPEPCK1 (grey bars) and AthPEPCK2 (white bars) by thermal shift assays coupled to differential scanning fluorometry.

Figure 3.
High-throughput screening of effectors binding to AthPEPCK1 (grey bars) and AthPEPCK2 (white bars) by thermal shift assays coupled to differential scanning fluorometry.

Experiments were performed in the presence of different concentrations of effectors (as indicated in the figure) and the shift in the melting temperature was calculated as described in the Experimental section. Data are the mean ± SE of three replicates for all samples and nine replicates for the control without ligand. • indicates a P-value < 0.05 and •• indicates a P-value < 0.01 using a t-test for two independent samples with a confidence level of 95%.

Figure 3.
High-throughput screening of effectors binding to AthPEPCK1 (grey bars) and AthPEPCK2 (white bars) by thermal shift assays coupled to differential scanning fluorometry.

Experiments were performed in the presence of different concentrations of effectors (as indicated in the figure) and the shift in the melting temperature was calculated as described in the Experimental section. Data are the mean ± SE of three replicates for all samples and nine replicates for the control without ligand. • indicates a P-value < 0.05 and •• indicates a P-value < 0.01 using a t-test for two independent samples with a confidence level of 95%.

Afterwards, we assessed the effect of the putative regulators on the decarboxylation activity of each enzyme (Figure 4 and Supplementary Figure S7). Glc6P, Fru6P and Glc1P inhibited AthPEPCK1 but not AthPEPCK2, whereas Fru1,6bisP inhibited both enzymes. In the case of AthPEPCK1, Glc6P produced the strongest inhibition (∼75%) at the highest concentration tested, whereas Glc1P, Fru6P and Fru1,6bisP produced ∼50% inhibition at the same concentration (Figure 4 and Supplementary Figure S7). For AthPEPCK1, the I0.5 values for Glc6P and Fru1,6bisP were 8.1 and 16 mM, respectively, whereas Glc1P and Fru6P exhibited I0.5 values ∼3-fold higher than Glc6P (Table 2).

Allosteric modulators of AthPEPCKs.

Figure 4.
Allosteric modulators of AthPEPCKs.

The activity of AthPEPCK1 (grey squares) and AthPEPCK2 (white squares) was measured at increasing concentrations of the metabolites indicated in the figure. Measurements were done as described in the Experimental section, in presence of 0.75 mM ATP and 0.75 mM OAA. The effect of malate was studied in the carboxylation direction at fixed substrate concentrations (0.13 mM ADP and 10 mM PEP) and variable concentrations of malate (squares) or malate-Mg2+ (triangles). Data are the mean of four independent data sets ± SE. Data were adjusted to a modified Hill equation (calculated parameters are shown in Table 2).

Figure 4.
Allosteric modulators of AthPEPCKs.

The activity of AthPEPCK1 (grey squares) and AthPEPCK2 (white squares) was measured at increasing concentrations of the metabolites indicated in the figure. Measurements were done as described in the Experimental section, in presence of 0.75 mM ATP and 0.75 mM OAA. The effect of malate was studied in the carboxylation direction at fixed substrate concentrations (0.13 mM ADP and 10 mM PEP) and variable concentrations of malate (squares) or malate-Mg2+ (triangles). Data are the mean of four independent data sets ± SE. Data were adjusted to a modified Hill equation (calculated parameters are shown in Table 2).

Table 2
Kinetic parameters for allosteric effectors of AthPEPCKs
Effector AthPEPCK1 AthPEPCK2 
I0.5 (mM) nH I0.5 (mM) nH 
Glc6P 8.1 ± 0.8 1.5 ± 0.2 N.E.b — 
Glc1P 26 ± 2 1.5 ± 0.2 N.E. — 
Fru6P 27 ± 2 0.9 ± 0.1 N.E. — 
Fru1,6bisP 16 ± 1 1.2 ± 0.1 43 ± 5 0.8 ± 0.1 
3PGA 21 ± 2 1.5 ± 0.2 35 ± 3 0.9 ± 0.1 
PPi 3.7 ± 0.1 1.5 ± 0.1 2.7 ± 0.4 1.1 ± 0.2 
Pyruvatea 15 ± 2 1.1 ± 0.2 27 ± 3 2.9 ± 0.8 
N-(phosphonomethyl)glycine 6.1 ± 0.6 0.8 ± 0.1 21 ± 4 0.9 ± 0.1 
α-ketoglutarate 13 ± 1 1.7 ± 0.4 41 ± 12 0.4 ± 0.1 
α-ketoglutarate-Mg2+ 19 ± 2 1.1 ± 0.1 N.E. — 
Fumarate 8.6 ± 0.5 1.7 ± 0.2 10 ± 1 1.8 ± 0.4 
Fumarate-Mg2+ 9.6 ± 0.7 1.9 ± 0.3 N.E. — 
Succinate 12.1 ± 0.4 1.4 ± 0.1 11 ± 1 0.9 ± 0.1 
Succinate-Mg2+ 14.5 ± 0.6 3.5 ± 0.6 18 ± 1 3.7 ± 0.9 
Citrate 2.1 ± 0.1 1.4 ± 0.1 3.7 ± 0.7 1.4 ± 0.4 
Citrate-Mg2+ 10.3 ± 0.6 1.5 ± 0.2 11.0 ± 0.6 2.3 ± 0.3 
Shikimate 10.4 ± 0.9 1.3 ± 0.1 N.E. — 
l-Glu 32 ± 5 0.9 ± 0.1 N.E. — 
UDP N.E. — 8 ± 1 0.8 ± 0.1 
 AthPEPCK1
 
AthPEPCK2
 
A0.5 (mM) nH A0.5 (mM) nH 
Malatea 2.6 ± 0.5 1.2 ± 0.3 N.E. — 
Malate-Mg2+a 2.1 ± 0.1 4.1 ± 0.8 N.E. — 
Effector AthPEPCK1 AthPEPCK2 
I0.5 (mM) nH I0.5 (mM) nH 
Glc6P 8.1 ± 0.8 1.5 ± 0.2 N.E.b — 
Glc1P 26 ± 2 1.5 ± 0.2 N.E. — 
Fru6P 27 ± 2 0.9 ± 0.1 N.E. — 
Fru1,6bisP 16 ± 1 1.2 ± 0.1 43 ± 5 0.8 ± 0.1 
3PGA 21 ± 2 1.5 ± 0.2 35 ± 3 0.9 ± 0.1 
PPi 3.7 ± 0.1 1.5 ± 0.1 2.7 ± 0.4 1.1 ± 0.2 
Pyruvatea 15 ± 2 1.1 ± 0.2 27 ± 3 2.9 ± 0.8 
N-(phosphonomethyl)glycine 6.1 ± 0.6 0.8 ± 0.1 21 ± 4 0.9 ± 0.1 
α-ketoglutarate 13 ± 1 1.7 ± 0.4 41 ± 12 0.4 ± 0.1 
α-ketoglutarate-Mg2+ 19 ± 2 1.1 ± 0.1 N.E. — 
Fumarate 8.6 ± 0.5 1.7 ± 0.2 10 ± 1 1.8 ± 0.4 
Fumarate-Mg2+ 9.6 ± 0.7 1.9 ± 0.3 N.E. — 
Succinate 12.1 ± 0.4 1.4 ± 0.1 11 ± 1 0.9 ± 0.1 
Succinate-Mg2+ 14.5 ± 0.6 3.5 ± 0.6 18 ± 1 3.7 ± 0.9 
Citrate 2.1 ± 0.1 1.4 ± 0.1 3.7 ± 0.7 1.4 ± 0.4 
Citrate-Mg2+ 10.3 ± 0.6 1.5 ± 0.2 11.0 ± 0.6 2.3 ± 0.3 
Shikimate 10.4 ± 0.9 1.3 ± 0.1 N.E. — 
l-Glu 32 ± 5 0.9 ± 0.1 N.E. — 
UDP N.E. — 8 ± 1 0.8 ± 0.1 
 AthPEPCK1
 
AthPEPCK2
 
A0.5 (mM) nH A0.5 (mM) nH 
Malatea 2.6 ± 0.5 1.2 ± 0.3 N.E. — 
Malate-Mg2+a 2.1 ± 0.1 4.1 ± 0.8 N.E. — 

Reactions were done as described in the Experimental section with fixed substrate concentrations of 0.75 mM ATP and 0.75 mM OAA. Constants were calculated by fitting experimental data presented in Figure 4 and Supplementary Figure S7 to the modified Hill equation.

a

To test the effect of malate and pyruvate, activity was measured in the carboxylation direction with substrate concentrations of 0.13 mM ADP and 10 mM PEP.

b

No effect was observed.

The organic acids anions α-ketoglutarate, fumarate, succinate and citrate displayed similar inhibition patterns for both enzymes, with fumarate showing the lowest I0.5 value (Table 2 and Supplementary Figure S7). To test whether the observed inhibition was due to free metal sequestration, we performed the same curves but preparing the organic acids with an equimolecular amount of Mg2+. Under the new conditions, the I0.5 values of AthPEPCK1 for α-ketoglutarate, fumarate and succinate were similar to those observed before adding Mg2+ (Table 2). Instead, the effect of these molecules on AthPEPCK2 became negligible, suggesting that inhibition was caused by free metal depletion. Similarly, further addition of Mg2+ reduced the inhibition caused by citrate, reflected by the increase in the I0.5 values (Table 2 and Supplementary Figure S7). Interestingly, malate activated AthPEPCK1 but had no effect on AthPEPCK2, while the presence of an equimolecular concentration of Mg2+ slightly increased the response of AthPEPCK1 to the activator (Figure 4 and Table 2). Shikimate and l-Glu also inhibited AthPEPCK1 but not AthPEPCK2 (Figure 4, Supplementary Figure S7 and Table 2). Considering all the effectors tested, PPi exerted the highest degree of inhibition (90%) and displayed the lowest I0.5 value (3.7 and 2.7 mM for AthPEPCK1 and AthPEPCK2, respectively; Figure 4 and Table 2). To rule out the possibility that PPi inhibition was caused by free metal sequestration, we performed PPi curves with equimolecular concentrations of Mg2+; under these conditions, the results were similar to those obtained without further additions of the divalent cation (data not shown).

Discussion

The properties of PEPCKs from photosynthetic organisms have been poorly explored in comparison with their counterparts from other organisms, with only a few reports on the literature (Supplementary Table S1). To expand our knowledge on the role of this enzyme in plant metabolism, we performed a detailed characterization of the kinetic and regulatory properties of the two PEPCKs present in Arabidopsis. One major hindrance for the study of source-derived PEPCK from C3, C4 and CAM species is its extreme sensitivity to proteolysis and multiple phosphorylation [11,14,46]. For these reasons, the use of recombinant enzymes is a convenient strategy to obtain homogeneous non-phosphorylated, non-proteolyzed enzyme preparations suitable for biochemical characterization [47,48]. This protocol should eliminate the discrepancies in the reported kinetic parameters (for example ∼6-fold differences in the Km for OAA in the case of Panicum maximum) due to different populations of the enzymes purified from a plant source [8,11,12,27,28,37,49].

We determined that recombinant AthPEPCKs are hexamers (Figure 1 and Supplementary Figure S2), the same quaternary structure reported for enzymes from C4 species (Urochloa panicoides, Chloris gayana and Panicum maximum) [8], but different from those reported for PEPCK from Cucurbita pepo [29,50] and a small version of the protein present in Anana comosus [11]. The hexameric structure has been recently confirmed in vivo in a paper published by Veyel et al. [41]. These authors reported that AthPEPCK1 possesses a native mass of 447 kDa in A. thaliana crude extracts, when analyzed by size exclusion chromatography coupled to protein identification by mass spectrometry (Supplementary material of reference [41]).

Traditionally, PEPCK activity was assayed with relatively high concentrations of Mn2+ (0.5–5 mM), conditions where the maximal activity is obtained (Figure 2A,C and Supplementary Table S1). However, as the concentration of this metal in plants cells remains within the micromolar range [35,37], in this work we characterized the recombinant enzymes from Arabidopsis using a physiological concentration of Mn2+ [37], to provide physiologically meaningful conclusions. Substrate kinetics determined under physiological conditions (4 mM MgCl2 plus 10 µM MnCl2) for AthPEPCKs are in good agreement with those reported for enzymes from other plant species (Table 1 and Supplementary Table S1). Considering that the reaction catalyzed by PEPCK is fully reversible in vitro, the higher apparent affinity for OAA and ATP exhibited by both recombinant enzymes (Table 1) is in accordance with the view that they primarily operate in the decarboxylation direction (gluconeogenic pathway) in vivo. Nevertheless, there might be circumstances when the carboxylation reaction is stimulated, as it has been recently described for an acetylated mammalian GTP-dependent PEPCK [51]. Strikingly, PEPCKs from C3 plants are relatively less active than the ones from C4 and CAM species (Table 1 and Supplementary Table S1). The activities of AthPEPCKs are similar to that from C. pepo, a C3 species (Supplementary Table S1). Conversely, PEPCKs from species performing C4 photosynthesis show ∼10-fold higher activity than AthPEPCKs (Supplementary Table S1). We speculate this is due to the key role that PEPCKs play in the CO2 concentrating mechanism operating in C4 species.

To test the ability of recombinant AthPEPCKs to bind putative effectors, we employed thermal shift assays coupled to differential scanning fluorometry. This technique was developed for high-throughput screening of ligands that might affect protein activity [52], becoming a fast, robust and economical technique to screen for enzyme effectors [4245]. The changes obtained in the Tm can be compared with those obtained by isothermal calorimetry and with IC50 values from enzymatic assays [42]. Using this method, several metabolites were identified as potential AthPEPCKs effectors. It is important to note that there was not a direct relationship between the increase in the melting temperature and the effect exerted by the ligand. For example, PPi was the most potent inhibitor of both AthPEPCKs, but it only increased the Tm of AthPEPCK1 and AthPEPCK2 by 0.7 and 0.8°C, respectively (Figures 3 and 4). Conversely, succinate displayed the highest Tm increase (5°C for AthPEPCK1 and 14°C for AthPEPCK2), but it only mediated a mild inhibition (Figure 3 and Supplementary Figure S6).

PEPCKs from the C4 plants U. panicoides and C. gayana are inhibited by 3PGA, Fru6P and Fru1,6bisP [8], as we observed for AthPEPCK1 (Table 2). On the other hand, the enzymes from these C4 plants were insensitive to l-Glu and malate, an inhibitor and an activator of AthPEPCK1, respectively [8,53]. In the case of the enzyme from C. pepo (a C3 plant), no effect was observed with succinate, malate and Fru1,6bisP [12], but this might be due to degradation of the enzyme as a consequence of the conditions employed in the purification [50]. A short version of a PEPCK present in the CAM plant A. comosus [11] is also inhibited by 3PGA and Fru1,6bisP, but succinate activated the pineapple enzyme while it inhibited AthPEPCKs. Also, malate had no effect on the enzyme of A. comosus although it activated AthPEPCK1 (Table 2).

PEP is involved in a complex metabolic network with multiple feedback controls [1]. It has been proposed that PEPCK activity is crucial during germination of oilseeds [2023,54]. Mutation of the PEPCK1 gene impairs the establishment of Arabidopsis seedlings [21,22]. We estimated that the cytosolic concentration of malate in Arabidopsis seedlings is close to 11 mM [55,56], a value considerably higher than the A0.5 of malate determined for AthPEPCK1 (Table 2). With this scenario in mind, we propose that activation of AthPEPCK1 by malate might be a mechanism to stimulate the flux of carbon released from fatty acids degradation through the glyoxylate cycle into gluconeogenesis (Figure 5A). This would allow the synthesis of sugars to feed the seedling until the development of the photosynthetic apparatus is complete.

Metabolic model of PEP metabolism in heterotrophic (A) and photoautotrophic (B) tissues.

Figure 5.
Metabolic model of PEP metabolism in heterotrophic (A) and photoautotrophic (B) tissues.

The enzymes involved in PEP metabolism are PEPCK, phosphoenolpyruvate carboxykinase (EC 4.1.1.49); PEPC, phosphoenolpyruvate carboxylase (EC 4.1.1.31); PPDK, pyruvate orthophosphate dikinase (EC 2.7.9.1); PK, pyruvate kinase (EC 2.7.1.40) and DHAP synthase, 3-deoxy-7-phosphoheptunolate synthase (EC 2.5.1.54). * Inhibition of glycolysis by PEP occurs trough inhibition of 6-phosphofructokinase (EC 2.7.1.11) and fructose-bisphosphate aldolase (EC 4.1.2.13) [9395,38].

Figure 5.
Metabolic model of PEP metabolism in heterotrophic (A) and photoautotrophic (B) tissues.

The enzymes involved in PEP metabolism are PEPCK, phosphoenolpyruvate carboxykinase (EC 4.1.1.49); PEPC, phosphoenolpyruvate carboxylase (EC 4.1.1.31); PPDK, pyruvate orthophosphate dikinase (EC 2.7.9.1); PK, pyruvate kinase (EC 2.7.1.40) and DHAP synthase, 3-deoxy-7-phosphoheptunolate synthase (EC 2.5.1.54). * Inhibition of glycolysis by PEP occurs trough inhibition of 6-phosphofructokinase (EC 2.7.1.11) and fructose-bisphosphate aldolase (EC 4.1.2.13) [9395,38].

Enzymes that catalyze opposite reactions and function in the same subcellular compartment are often regulated in a reciprocal way to avoid futile cycles [1,57]. Such cycle would occur by simultaneous operation of PEPCK and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) which, if not regulated, would cause the net hydrolysis of ATP to ADP and Pi. Malate activates AthPEPCK1 in the direction of PEP synthesis (Table 2) and, at the same time, it inhibits PEPC, which uses PEP [5864], providing the necessary regulation (Figure 5A). As previously mentioned, plant PEPCKs are phosphorylated [29,46,6569]. Leegood and Walker [67] proposed that this mechanism might coordinate the activities of both enzymes. In leaves of C4 and CAM plants, light promotes the dephosphorylation of PEPC (reducing its activity and making it less sensitive to activators and more sensitive to inhibitors) and PEPCK (which increases its activity). Mass spectrometry data available in the PhosPhat database [70] show that both AthPEPCK1 and AthPEPCK2 are subject to phosphorylation on multiple sites (Supplementary Figure S1). Nevertheless, the effects on kinetic parameters or the tissues and physiological conditions where phosphorylation occurs remain to be elucidated.

A second futile cycle could occur between PEPCK and pyruvate kinase (PK, EC 2.7.1.40), causing the net decarboxylation of OAA to Pyr and CO2 [1]. In germinating oilseeds two isoenzymes of PK are expressed, one in the plastid and the other one in the cytosol, which accounts for 85% of the total PK activity [71,72]. As with PEPC, malate inhibition of PK [73] would avoid a futile cycle between these enzymes (Figure 5A).

Recently, Ferjani et al. [74] proposed a regulatory role for PPi in gluconeogenesis during seed germination. The role of this metabolite in plant metabolism remained elusive for years [75,76], but these authors reported that knock-out mutants in the vacuolar H+-translocating pyrophosphatase (fugu5 mutant) accumulate high levels of PPi in the cytosol, inhibiting the gluconeogenic pathway and arresting the development of Arabidopsis seedlings [75]. The inhibition of gluconeogenesis has been related to product inhibition of UDP-glucose pyrophosphorylase [74], which is strongly inhibited by PPi concentrations lower than 10 mM [77]. As AthPEPCKs are strongly inhibited by PPi (Figure 4 and Table 2), we hypothesize that inhibition of gluconeogenesis might also be the consequence of AthPEPCKs inhibition (Figure 5A).

In other non-photosynthetic tissues, such as fruits, enzymatic control by metabolites could also be of importance. Both malate levels and PEPCK activity peak during tomato and strawberry fruit ripening [26,7881]. The suppression of the PEPCK gene by RNA interference in Solanoum tuberosum led to a reduced sugar content accompanied by an increase in malate levels [82,83]. The opposite effect was observed when this gene was overexpressed with the 35S promoter or with a fruit specific E8 promoter [84]. Thus, activation of AthPEPCK1 by malate might be a mechanism to channel malate towards gluconeogenesis. Furthermore, the relationship between AthPEPCK1 and malate might be important during stress conditions. Arabidopsis pepck1 knock-out mutants are more susceptible to drought than wild type plants. Mutant plants are unable to completely close their stomata in the dark [85], which is closely related to malate metabolism [86]. Recently, this relationship has been clearly established as pepck1 mutants have increased levels of malate, succinate and citrate, and reduced levels of Glc1P, Glc6P and Fru6P [74].

The development of the photosynthetic apparatus leads to a major rearrangement of the metabolic profile of the plant cell [87]. The main carbon source in illuminated leaves comes from trioses-P synthesized by the Benson–Calvin cycle [39,88]. Under this metabolic scenario, there is an exponential increase in the levels of trioses-P and hexoses-P (Figure 5B). We estimated that the concentrations of hexoses-P in illuminated leaves are within the range of the I0.5 values determined for AthPEPCKs (Table 2) [55,87], which are expressed in this tissue [19]. Then, hexoses-P would inhibit PEPCKs and stimulate the activity of PEPC, which is activated by Glc6P [5864], thus inhibiting gluconeogenesis and stimulating the respiration of the photosynthetically fixed carbon (Figure 5B).

Another function of AthPEPCKs in leaves might be in stress-related metabolic responses. Shikimate metabolism in plants is an intricated network of enzymatic reactions dedicated to the synthesis of the aromatic amino acids (Phe, Trp, Tyr) and a range of metabolic compounds, some of which are related to biotic stress reactions, such as salicylic acid [89]. This pathway takes place mainly in the plastids and starts with the condensation of PEP (derived from glycolysis) with eritrose-4-phosphate (E4P, derived from the pentose phosphate pathway) to form shikimate, the precursor of aromatic amino acids in the plastids and other compounds in the cytosol [89]. Inhibition of AthPEPCK1 by shikimate might be a mechanism to regulate the synthesis of PEP, the initial substrate of the shikimate pathway. It has been described that partitioning of carbon through this pathway is feedback inhibited by the corresponding products of each branch and supply of the initial substrates of the pathway (PEP and E4P) play an important role in the flux through it [89]. The calculated concentration of shikimate in Arabidopsis leaves (1.1 mM) [55,87] is in the range of the I0.5 obtained for AthPEPCK1. This relation of AthPEPCKs with the shikimate pathway is reinforced by the fact that in Arabidopsis, the PEPCK1 gene is strongly induced in leaves upon biotic stress (as it can be seen using data from the gene expression database eFP Browser [90]). Also, mutants in the PEPCK1 gene show reduced levels of aromatic amino acids in etiolated Arabidopsis seedlings [74] and, in pepper plants where the PEPCK gene was silenced the levels of salicylic acid were reduced compared with the wt upon bacterial infection [91]. Both aromatic amino acids and salicylic acid represent products of the shikimate pathway [89,92].

The differences found in the regulatory properties of plant PEPCKs (this and further works detailed in Supplementary Table S1) might result from variants in the metabolic pathways operating in diverse plant species. Another possibility is that the kinetic and regulatory properties determined for native PEPCKs purified directly from a plant tissue reflect the response of a mix of phosphorylated (or proteolyzed) and non-phosphorylated enzyme. The development of a system for PEPCK expression and purification will allow us to mimic such modifications by mutagenesis in the near future, to better understand the regulation of this pivotal enzyme.

Abbreviations

     
  • 3PGA

    3-phosphoglycerate

  •  
  • E4P

    erythrose-4-phosphate

  •  
  • Fru6P

    fructose 6-phosphate

  •  
  • Fructose 1,6-bisphosphate

    Fru1,6bisP

  •  
  • Glc1P

    glucose 1-phosphate

  •  
  • Glc6P

    glucose 6-phosphate

  •  
  • OAA

    oxaloacetic acid

  •  
  • PEP

    phosphoenolpyruvate

  •  
  • PEPC

    phosphoenolpyruvate carboxylase

  •  
  • PEPCK

    phosphoenolpyruvate carboxykinase

  •  
  • PK

    pyruvate kinase

  •  
  • Pyr

    pyruvate

Author Contributions

F.E.P. and A.A.I. conceived the study. B.E.R., M.D.H. and L.L. performed experiments. B.E.R., M.D.H., C.M.F. and A.A.I. analyzed data and wrote the manuscript. All authors approved the final version of the manuscript.

Funding

This work was supported by grants from ANPCyT (PICT-2015-1767, PICT-2015-1074, PICT-2017-1515), CONICET and UNL (CAI+D 2016, CAI+D Joven 2016).

Acknowledgements

BER is a Fellow from CONICET; CMF, FEP and AAI are Researchers from the same Institution. CMF is funded by the Max Planck Society (Partner Group for Plant Biochemistry).

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

Current address: Max Planck Institute for Biology of Ageing, Cologne, Germany.

Current address: Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.