Protein Expression and Purification 110 (2015) 7–13

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Expression, purification and characterization of Solanum tuberosum recombinant cytosolic pyruvate kinase Evgenia L. Auslender, Sonia Dorion, Sébastien Dumont, Jean Rivoal ⇑ Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 Sherbrooke Est, Montréal, Qc H1X 2B2, Canada

a r t i c l e

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Article history: Received 24 September 2014 and in revised form 19 December 2014 Available online 5 January 2015 Keywords: Plant Pyruvate kinase Respiratory metabolism Glycolysis Recombinant protein Chaperone

a b s t r a c t The cDNA encoding for a Solanum tuberosum cytosolic pyruvate kinase 1 (PKc1) highly expressed in tuber tissue was cloned in the bacterial expression vector pProEX HTc. The construct carried a hexahistidine tag in N-terminal position to facilitate purification of the recombinant protein. Production of high levels of soluble recombinant PKc1 in Escherichia coli was only possible when using a co-expression strategy with the chaperones GroES-GroEL. Purification of the protein by Ni2 + chelation chromatography yielded a single protein with an apparent molecular mass of 58 kDa and a specific activity of 34 units mg1 protein. The recombinant enzyme had an optimum pH between 6 and 7. It was relatively heat stable as it retained 80% of its activity after 2 min at 75 °C. Hyperbolic saturation kinetics were observed with ADP and UDP whereas sigmoidal saturation was observed during analysis of phosphoenolpyruvate binding. Among possible effectors tested, aspartate and glutamate had no effect on enzyme activity, whereas a-ketoglutarate and citrate were the most potent inhibitors. When tested on phosphoenolpyruvate saturation kinetics, these latter compounds increased S0.5. These findings suggest that S. tuberosum PKc1 is subject to a strong control by respiratory metabolism exerted via citrate and other tricarboxylic acid cycle intermediates. Ó 2014 Elsevier Inc. All rights reserved.

Introduction Pyruvate kinase (PK1, EC 2.7.1.40) catalyzes the irreversible transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, thereby generating pyruvate and ATP. It is therefore involved in the provision of pyruvate used as substrate for aerobic respiration, the substrate-level synthesis of ATP and in the control of the metabolism of the important glycolytic intermediate PEP. In plants, PK exists as tissue-specific isozymes that are present in the cytosol (PKc) and the plastid (PKp) [1]. Plant PKs are encoded by a relatively large gene family. For instance, the genome of the model plant Arabidopsis thaliana contains 10 PKc genes, 3 PKp genes and 1 PK-like gene [2,3]. In Solanum tuberosum, 5 PKcs and 4 PKps have been identified [3]. In plants, PKc is important in sink-source relationship as well as for the control of carbon and respiratory metabolism [3–5]. PKp activity is needed for the catabolism of storage compounds in germinating seeds [6] and the biosynthesis of seed oil [2]. The kinetic ⇑ Corresponding author. Tel.: +1 514 343 2150; fax: +1 514 343 2288. E-mail address: [email protected] (J. Rivoal). Abbreviations used: DTT, dithiothreitol; IPTG, isopropyl b-D-thiogalactoside; LDH, lactate dehydrogenase; Ni–NTA, Ni2+–nitrilotriacetic acid; PEP, phosphoenolpyruvate; PEG, polyethylene glycol; PK, pyruvate kinase; PKc, cytosolic pyruvate kinase; PKp, plastidic pyruvate kinase; TCA, tricarboxylic acid. 1

http://dx.doi.org/10.1016/j.pep.2014.12.015 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.

properties of native plant PKs purified from a variety of sources have been the subject of extensive characterization. From these studies, it appears that the regulatory properties of PKs isozymes are often species-, tissue- and developmental stage-specific, suggesting a high level of specialization in the regulation of PKs depending on particular organismal and metabolic contexts [1]. Plant PKc allosteric regulation by amino acids Asp and Glu as well as intermediates of the glycolytic pathway and the tricarboxylic acid (TCA) cycle was demonstrated in a number of cases [7–9]. Strong sensitivity of the enzyme to pH has also been reported [7–9]. Additional PKc regulatory mechanisms have been described [10]. These include evidence for the involvement of proteolytic processing at the C-terminus as well as phosphorylation and ubiquitination in the regulation of PKc activity and in vivo steady-state levels. However, the importance of posttranslational modifications in the control of PKc is still not completely elucidated. Further studies of the enzymes involved in PKc phosphorylation and proteolytic processing would benefit from the availability of the enzyme in recombinant form. However, despite the general importance of PKs in plant central metabolism, only two reports describe the successful expression of plant PKs in heterologous systems and little information is available on the properties of the recombinant enzymes [2,10]. We wanted to expand the range of available recombinant PKs by attempting the production of high yields of recombinant PKc as this would greatly facilitate fur-

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ther studies of this enzyme, in particular the molecular characterization of protein kinase(s) and the proteolytic process putatively involved in posttranslational regulation of this enzyme [10]. The primary goal of the present study was therefore the purification and the characterization of a recombinant PKc produced in Escherichia coli. To this end, we used the sequence of the first cloned PKc cDNA [11]. This sequence is encoded by the S. tuberosum (potato) PKCYT1 gene, which appears to be highly expressed in tubers [3]. The production of high yields of the recombinant protein in E. coli was only possible with the co-expression of the GroES-GroEL chaperone complex encoded by the pGro7 plasmid. We report on some of the kinetic properties of the recombinant enzyme, including substrate saturation behavior and identify several intermediates of the TCA cycle as effectors. Materials and methods Materials and chemicals Buffers, chemicals, metabolites and reagents used were of analytical grade and purchased from Sigma Chemical (St. Louis, MO) or Fisher Scientific (Nepean, ON, Canada). Restriction enzymes and reagents used for recombinant DNA work were purchased from Fermentas (Burlington, ON, Canada). The pGro7 plasmid was from Takara Bio Inc. (Mountain View, CA). Sequencing primers were from Sigma Genosys (The Woodlands, TX). The pProEX HTc expression vector and Ni2+–nitrilotriacetic acid (Ni–NTA) agarose were supplied by Invitrogen (Burlington, ON, Canada).

adjusted to pH 8.0 with NaOH. Cells were disrupted at 4 °C twice using a French press (18,000 psi) and the resulting extract was centrifuged for 30 min at 10,000g. The supernatant was filtered over a cellulose acetate filter (0.22 lm pore size). At this step, in some experiments, aliquots of the pellet and the supernatant were subjected to electrophoretic analysis. To purify soluble (6xHis)StPKc1, the supernatant was incubated with Ni–NTA resin (0.5 mL settled bed volume) previously equilibrated in extraction buffer. The protein was allowed to bind to the resin for 1 h at 4 °C. Subsequent steps in the purification were performed at room temperature. The slurry was then poured into a 0.5 cm-diameter column. All the extract was allowed to pass through the column. Subsequently, the column was washed using 30 volumes of new extraction buffer followed by 60 volumes of extraction buffer containing 20 mM imidazole. The column was then washed with 60 volumes of a solution buffer containing 50 mM NaH2PO4, 5 mM ATP, 10 mM MgCl2 and 300 mM KCl, adjusted to pH 8.0 with NaOH (ATP wash buffer). Elution of the bound proteins from the column was achieved with 50 mM NaH2PO4, 300 mM NaCl and 250 mM imidazole, adjusted to pH 8.0 with NaOH. Ten 0.5 mL fractions were collected. Fractions containing the peak of (6xHis)StPKc1 (typically 1.5 mL total) were identified by SDS–PAGE stained by Coomassie blue and activity assays. These fractions were dialyzed against a buffer containing 50 mM Tris–Cl, pH 7.5 and 1 mM dithiotreitol (DTT). The dialyzed enzyme preparation was centrifuged for 10 min at 10,000g and the supernatant stored at 20 °C in 50% (v/v) glycerol until used. Pyruvate kinase activity assay and protein determination

S. tuberosum PKc cDNA in pBluescript SK () (StPKc) [11] was digested with MscI and SacI, producing a 1577 bp DNA fragment with a blunt 50 end and cohesive 30 extremity. The 50 extremity of this fragment corresponds to the fourth nucleotide of the PKc coding sequence. The fragment also contained the stop codon of the StPKc sequence. This fragment was cloned into the pProEX HTc expression vector previously digested with StuI and SacI. The generated construct encoded a 541 amino acid recombinant protein with a theoretical molecular weight of 58,922.3 Da. The N-terminal extension of its sequence contained a hexahistidine (6xHis) tag and the encoded protein was named (6xHis)StPKc1. The ligated plasmid was used to transform different competent E. coli cells (strains DH5a, BL21(DE3), Rosetta™(DE3), HB101 and HB101 carrying the pGro7 plasmid encoding for GroES-GroEL) in various attempts to optimize production of recombinant protein in the soluble fraction. The identity of the construct was confirmed by diagnostic digestions and sequencing.

To assay recombinant PKc activity, the PK reaction was coupled to that of lactate dehydrogenase (LDH, EC 1.1.1.27) [12]. Spectrophotometric assays were carried out at 30 °C and monitored at 340 nm using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA). One unit (U) of activity corresponds to the production of 1 lmol of pyruvate per minute. Except when mentioned otherwise, reactions were done in a solution containing 50 mM Tris–HCl pH 7.5, 10 mM MgCl2, 50 mM KCl, 4 mM PEP, 4 mM MgADP, 0.32 mM NADH and 0.4 U LDH in a final volume of 200 lL. Reaction rates were linear with time and proportional to the quantity of (6xHis)StPKc added. Enzyme activity measurements at different pHs during the determination of optimum pH, substrate saturation kinetic and effector studies were done by substituting the 50 mM Tris buffer in the assay described above by a buffer capable of maintaining ionic strength (0.05 M acetic acid, 0.05 M 2-(Nmorpholino)ethanesulfonic acid and 0.1 M Tris) [13]. In this case, pH was adjusted by adding NaOH or HCl. Enzyme assays and kinetic analyses were carried out in triplicate on a minimum of two independent enzyme preparations. Protein was determined using the method of Bradford [14] with bovine serum albumin as a standard.

Production of (6xHis)StPKc1

Electrophoresis and immunoblot analysis

E. coli carrying the construct encoding (6xHis)StPKc1 was grown in Luria–Bertani broth medium (200 mL total volume) at 37 °C to an A600 of 0.5 with the appropriate antibiotics. Isopropyl ß-D-thiogalactoside (IPTG) was then added to the culture at a final concentration of 0.6 mM and bacteria were grown with agitation (250 rpm) for 18 h at 18 °C. For the production of control (non-induced) cultures, the cultures were grown similarly, but in absence of IPTG. For the production of (6xHis)StPKc1 in HB101 carrying the pGro7 plasmid, cells were grown and induced as described above, except that they were inoculated in the presence of L-arabinose (0.5 mg mL1). Cells were centrifuged for 10 min at 5000g at 4 °C. Cell pellets were resuspended in 10 mL of extraction buffer containing 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 0.1% (w/v) Triton X-100, 1 mM phenylmethylsulphonylfluoride,

Analysis of proteins by electrophoresis in polyacrylamide gels under denaturing conditions in the presence of SDS (SDS–PAGE) was performed using 10% (w/v) acrylamide gels. Gels were stained with Coomassie R-250 or transferred to nitrocellulose [15]. Immunoblot analysis of PKc was performed with anti-PKc polyclonal immune serum (1/5000 dilution) directed against the Brassica napus native enzyme [7]. The polyhistidine tag was detected using the PentaHis mouse monoclonal antibody (1/2000 dilution) from Qiagen (Toronto, ON, Canada). The GroEL protein was detected using the anti-E. coli GroEL mouse monoclonal antibody clone 9A1/2 (1/10,000 dilution) from One World Lab (San Diego, CA). Detection of antigen/antibody complexes was done using alkaline phosphatase-tagged secondary antibodies (Promega, Nepean, ON, Canada) [15].

Construction for recombinant PKc expression in E. coli

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Characterization of (6xHis)StPKc1 sensitivity to temperature and cosolvent The heat stability of (6xHis)StPKc1 was measured as follows: Sixty microliter aliquots of the purified protein in a solution containing 25 mM Tris–Cl, pH 8.0, 0.5 mM DTT and 50% (v/v) glycerol were incubated at various temperatures in Eppendorf tubes for exactly 2 min using dry baths. The tubes were then transferred to an ice bath for 1 min. The remaining enzyme activity was then immediately assayed as described above using 10 lL aliquots of the heat-treated sample. The effect of co-solute on the enzyme activity was measured by adding glycerol (final concentration ranging from 5% to 20% (v/v)) or polyethylene glycol 8000 (PEG) (final concentration ranging from 0% to 20% (v/v)) to the reaction mixture. Characterization of (6xHis)StPKc1 kinetic properties Substrate saturation kinetics were carried out at different reaction pHs for PEP, MgADP and MgUDP by modifying concentrations of one substrate at a time. In the reactions carried out with UDP, ADP was omitted from the reaction mixture. Apparent Km for

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ADP and UDP substrates were calculated from the Michaelis–Menten equation using SigmaPlot 8.0 (SPSS, Chicago, IL). S0.5 and h for PEP were calculated using a previously described kinetics program [16]. Effects of glutamate, aspartate, succinate, a-ketoglutarate, isocitrate and citrate were evaluated on the PEP saturation curve of the enzyme and at different reaction pHs. Results and discussion Optimization of the production and purification of (6xHis)StPKc1 in soluble form Initial trials to produce (6xHis)StPKc1 in E. coli BL21(DE3) strain resulted in the production of large amounts of recombinant protein in insoluble form, indicating that (6xHis)StPKc1 was mainly produced in inclusion bodies. Additional production trials yielded similar results with E. coli strains DH5a, Rosetta™(DE3) and HB101. Varying induction time, IPTG concentration or temperature during growth did not improve these results. Fig. 1A shows an example of (6xHis)StPKc1 induction carried out in E. coli HB101. PK activity detected in the supernatant of broken cells probably reflected endogenous E. coli PK because it was not significantly dif-

Fig. 1. Optimization of the production and purification of soluble (6xHis)StPKc1. (A) SDS–PAGE analysis of E. coli HB101 carrying (6xHis)StPKc1 in pProEX HTc. Total cell lysate (T), insoluble (I) and soluble (S) fractions corresponding to 60 lL of culture were prepared from cells grown in the absence () or the presence (+) of IPTG. (B) SDS–PAGE analysis of E. coli HB101 carrying pProEX HTc encoding (6xHis)StPKc1 and pGro7 encoding GroEL–GroES. Cells were grown with L-arabinose in absence () or the presence (+) of IPTG. Lanes were loaded and identified as in (A). (C) Immunoblot analysis of a gel identical to (B) transferred to nitrocellulose and probed with the PentaHis mouse monoclonal antibody. (D) Immunoblot analysis of a gel identical to (B) transferred to nitrocellulose and probed with a mouse monoclonal antibody directed against E. coli GroEL. (E) PK specific activity in the soluble fractions of extracts shown in (A) and (B). (F) SDS–PAGE analysis of (6xHis)StPKc1 purified from the soluble fraction of E. coli HB101 co-expressing GroEL–GroES as in (B). Purification was done using chromatography on Ni–NTA resin with an ATP wash step as described in Materials and methods. A 0.5 lg aliquot of the purified protein was loaded on lane 1 and protein staining was done with Coomassie brilliant blue R-250. (G) Immunoblot analysis of the purified protein (50 ng) using an anti-B. napus PKc polyclonal antibody. (H) Immunoblot analysis of the purified protein (50 ng) using the PentaHis monoclonal antibody. For all gels and blots, the running position of molecular weight markers (M) is indicated on the left.

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Table 1 Purification of (6xHis)StPKc1 from 200 mL of bacterial culture (E. coli HB101 co-expressing the GroEL–GroES chaperone complex). Step Crude extract Dialyzed Ni–NTA eluate a

Total activity (U) 72.15 23.98

a

Total protein (mg)

Specific activity (U mg1 protein)

Yield (%)

Purification (fold)

54.25 0.69

1.33 34.76

100 33.24

 26.14

The crude extract also contains endogenous bacterial PK activity.

ferent in the presence or absence of IPTG (Fig. 1E). The production of large amounts of recombinant protein was therefore not feasible under these conditions. We consequently sought to increase the yields of soluble (6xHis)StPKc1. Attempts at solubilizing active recombinant protein from the pellet were not successful (data not shown). The problem of insoluble protein accumulation during heterologous expression in E. coli of large multidomain proteins such as (6xHis)StPKc1 is a common issue associated with protein misfolding [17]. The use of folding helpers, including molecular chaperones has often proven useful in solving this issue [18–20]. For these reasons, we opted to induce the production of (6xHis)StPKc1 in the presence of the GroES-GroEL chaperone complex encoded by the pGro7 plasmid and under the control of the Larabinose-inducible araB promoter. SDS–PAGE analysis of equal volumes of E. coli HB101 culture grown in the presence of L-arabinose and with or without IPTG was performed (Fig. 1B). The results indicate a lesser total protein content in the culture growing in the presence of IPTG. This may reflect the reported negative effect of IPTG on bacterial growth [21]. However, in E. coli HB101 strain co-expressing (6xHis)StPKc1 and the GroES-GroEL chaperone complex, specific PK activity was increased by 4.3-fold in the supernatant of cells induced by IPTG compared to non-induced cells (Fig. 1E). SDS–PAGE analysis of cells grown in the absence and the presence of IPTG revealed a band at approximately 58 kDa (Fig. 1B), which corresponds to the predicted Mr of both (6xHis)StPKc1 and GroEL. Indeed, the GroEL chaperone (HSP60) has approximately the same molecular mass as (6xHis)StPKc1 [22]. We carried out immunoblot analysis of extracts of E. coli HB101 strain co-expressing (6xHis)StPKc1 and the GroES-GroEL chaperone complex using the PentaHis mouse monoclonal antibody (Fig. 1C) and a mouse monoclonal antibody directed against E. coli GroEL (Fig. 1D). A 58 kDa polyhistidine tagged protein could be detected in IPTG-induced bacterial extracts, and a significant fraction of this protein was soluble (Fig. 1C). The GroEL chaperone was detected in the presence and absence of IPTG and migrated at the same Mr as the polyhistidine tagged protein (Fig. 1D). It was therefore difficult to differentiate GroEL from (6xHis)StPKc1 by SDS–PAGE. The GroEL protein does not carry a (6xHis) tag and was consequently not expected to bind to the Ni–NTA resin. Nevertheless, when (6xHis)StPKc1 was purified without the inclusion of the ATP wash buffer step in the purification protocol, the GroEL protein could be detected together with (6xHis)StPKc1 in the eluate from the Ni–NTA affinity column (Fig. S1). The addition of the ATP wash buffer step proved necessary to completely remove traces of GroEL from the purified (6xHis)StPKc1 (Fig. S1). Such requirement for a buffer containing ATP, MgCl2 and KCl was previously shown to be useful for the removal of co-purifying GroEL chaperone in the production of recombinant Interleukin-2 Tyrosine kinase [23]. To purify (6xHis)StPKc1, we therefore prepared an extract from bacterial cell cultures co-expressing the (6xHis)StPKc1 and the GroES-GroEL chaperone complex. The soluble proteins were subjected to metal affinity chromatography on Ni–NTA resin. This resin was subjected to an ATP wash buffer step which was followed by elution of the bound protein using 250 mM imidazole. The purified protein was subjected to SDS– PAGE (Fig. 1F). A single protein band with a molecular weight of 58 kDa was detected in the eluate, demonstrating electrophoretic homogeneity. The purified protein was recognized by a polyclonal

antibody raised against native B. napus PKc (Fig. 1G) and by the PentaHis monoclonal antibody (Fig. 1H), confirming that the purified protein was a polyhistidine tagged PK. As mentioned above, the protein was free from GroEL contamination (Fig. S1). The PK specific activity of the purified fraction was 34.76 U mg1 protein (Table 1). This value is comparable to reported specific activity values for purified native plant PKcs which usually lie in the range of 20–73 U mg1 protein [7,9,24].

Characterization of (6xHis)StPKc1 properties Effect of pH The activity of (6xHis)StPKc1 was assayed as a function of pH. The enzyme retained more than 80% of its maximum activity between pH 6 and 7.5 (Fig. 2A). A rapid decrease in activity was observed at pH 8 and higher values. Similar slightly acidic pH optima have been observed for purified native plant PKcs [7,8,25,26]. This feature contrasts with the generally alkaline pH optima observed for native plant PKps [26,27]. Since the PKCYT1 gene is

Fig. 2. Effect of pH and temperature on (6xHis)StPKc1 activity. (A) Dependence of activity on assay pH. Values are representative of data obtained from two different purifications. Activity measured at pH 6.4 (0.16 mU) represents 100%. (B) Thermal stability of (6xHis)StPKc1. Values are representative of data obtained from three different purifications. Activity measured after incubation at 40 °C (0.26 mU) represents 100%. Enzyme was incubated at a given temperature for 2 min as described under Materials and methods. Activity was assayed at 30 °C.

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expressed in the tuber, it is relevant to relate this property to the physiological conditions existing in this organ. Indeed, the potato tuber is known to be a hypoxic organ and the pH of the cytosol of plant cells under O2 shortage is often slightly acidic [28,29]. Thus, prevailing pH conditions in the tuber, would not significantly restrict the activity of product of the PKCYT1 gene. Thermal stability A characteristic shared by many plant PKcs, but not PKps, is its relative stability upon thermal denaturation [24,25,27]. This property is sometimes used in purification schemes of native plant PKcs [9,30]. In keeping with this attribute of PKcs, (6xHis)StPKc1 demonstrated a remarkable stability at high temperatures, conserving 80% of its activity after 2 min at 75 °C (Fig. 2B). Complete denaturation occurred sharply at higher temperatures and less than 10% of activity remained after treatment at 85 °C. Organic co-solute effect on activity It was previously observed [31] that organic co-solutes such as PEG had a stimulatory effect on PKc activity (e.g. 2-fold increase in specific activity in the presence of 5% (v/v) PEG. PEG was found to promote the association of the subunits of the enzyme purified from Ricinus communis into its active tetrameric form [31]. Such significant effect of the presence of organic co-solutes on purified (6xHis)StPKc1 activity was not observed. Only very modest (up to 15%) increases in specific activity could be observed with the addition of glycerol or PEG in the range of 5–10% (v/v) in the assay mixture (data not shown). Saturation kinetics with PEP and nucleoside diphosphate substrates The effect of varying assay pH in the physiological range (pH 6.5–7.5) on the binding of PEP, MgADP and MgUDP to (6xHis)StPKc1 was examined. The apparent Km or S0.5, Vmax and Vmax/Km or Vmax/S0.5 values were calculated (Fig. 3). In the case of nucleoside diphosphate substrates, hyperbolic behavior was detected, similarly to observations made with PKcs purified from R. communis leaves [25] and B. napus suspension cell cultures [7]. Apparent Km values of (6xHis)StPKc1 for ADP (Fig. 3A) were generally higher than those reported for native plant PKcs, which lie in the range of 0.05–0.09 mM [9]. This perhaps reflects a difference between native PKcs and the heterologously expressed enzyme. Under these latter conditions, protein folding or posttranslational modifications that could occur in planta may not take place. It is however possible that low affinity for MgADP represents a distinct feature of the enzyme present in the potato tuber which is a specialized and physiologically distinct reserve organ. Affinity of PKcs for MgUDP has less frequently been reported in the literature. Again, our recombinant enzyme had high apparent Km values for MgUDP compared to other published values (e.g. 0.16 mM in B. napus [7] and 0.7 mM in spinach leaves [32]). For MgADP, pH had only slight effect on Km. In the case of MgUDP, the effect of pH on affinity was more prominent. In both cases, the lowest Km values were obtained at pH 6.8. When PEP saturation kinetics were examined, a positive cooperative binding was observed (see data below). Affinity of the recombinant protein for this substrate was significantly lower than reported values for purified native PKcs which usually lie below 0.2 mM [7–9,25] in the pH range tested here. Affinity of (6xHis)StPKc1 for PEP increased with greater pH values, as previously observed [7,9,25]. If the affinity of (6xHis)StPKc1 reflects that of the native potato enzyme, it is likely that reported concentration of substrates in the cytosolic compartment [33] greatly limits the activity of the enzyme. Maximal velocity of the enzyme was negatively affected by increasing pH (Fig. 3B), in accordance with previous reports for native PKcs [7,9,25]. A relative insensitivity of (6xHis)StPKc1’s catalytic efficiency (Vmax/Km or Vmax/S0.5) to pH in the range 6.5–7.5 was

Fig. 3. Effect of pH on substrate saturation kinetics (A) Values of apparent Km (for ADP and UDP) and S0.5 (PEP) are plotted against assay pH. (B) Apparent maximal velocity of the enzyme plotted against assay pH. (C) Catalytic efficiency calculated as Km/Vmax (for ADP and UDP) or S0.5/Vmax (for PEP) plotted against assay pH. Nucleoside diphosphates were prepared as Mg salts.

observed with all substrates tested (Fig. 3C). Although, as usually observed with PKs, catalytic efficiencies with MgADP were always higher than with MgUDP, the lack of effect of pH contrasts with previous observations where this parameter was slightly [7,9] to strongly [25] positively affected by increasing assay pH in the same range. Metabolite effects on PEP binding A number of amino and organic acids have been implicated in the metabolic regulation of PKcs in various plant tissues. We therefore tested several of these metabolites at various pHs for their effect on (6xHis)StPKc1 activity (Table 2). Amino acids Asp and Glu did not significantly affect the activity of the enzyme. This situation contrasts with previous reports that provide evidence for allosteric regulation by amino acids, in particular by Asp and Glu. Indeed, Glu is a potent inhibitor of a number of purified native

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Table 2 Effect of metabolites on (6xHis)StPKc1 activity tested at various pHs. Effector concentration in these assays was 25 mM. Metabolite

Relative activity (%)

Aspartate Glutamate Citrate Isocitrate a-Ketoglutarate Succinate

pH 6.5

pH 6.8

pH 7.5

103.9 ± 7.9 97.3 ± 4.8 2.2 ± 1.3 62.8 ± 7.4 46.4 ± 17.3 89.5 ± 9.1

92.5 ± 5.8 90.3 ± 4.8 4.3 ± 1.3 61.3 ± 3.8 65.6 ± 7.2 66.3 ± 8.3

113.2 ± 8.9 107.3 ± 11.8 12.5 ± 3.4 91.5 ± 10.8 65.0 ± 3.6 98.8 ± 6.7

Table 3 Effect of a-ketoglutarate and citrate at various pHs on affinity of (6xHis)StPKc1 for PEP. Effector

Concentration (mM)

None

a-Ketoglutarate 10

Citrate

15 25 5 10

S0.5 (mM)

results therefore provide evidence for an absence of regulation of (6xHis)StPKc1 by amino acids and a relatively important inhibitory effect by intermediates of the TCA cycle. These data could indicate that activity of the product of the PKCYT1 gene in tubers is mainly controlled by respiratory metabolism and that metabolic feedback occurs by sensing of altered levels in TCA cycle intermediates, principally citrate. Variations in citrate could result in modulation in TCA cycle activity. Several enzymes of the TCA cycle are sensitive to inhibition by reduced pyridine nucleotides [34], in particular isocitrate dehydrogenase [35]. An elevation of NAD(P)H/NAD(P) ratios in mitochondria, could therefore inactivate isocitrate dehydrogenase, thereby slowing down citrate consumption and increasing its levels. This situation would in turn inhibit PKc, thus restricting the flux of carbon directed to pyruvate and mitochondrial respiration. Conclusions

pH 6.5

pH 6.8

pH 7.5

3.38 ± 0.59 4.01 ± 0.28 4.61 ± 0.66 6.26 ± 0.53 3.35 ± 0.38 5.05 ± 0.47

2.99 ± 0.54 3.04 ± 0.26 3.39 ± 0.38 4.23 ± 0.26 3.45 ± 0.62 4.89 ± 1.05

1.96 ± 0.12 2.34 ± 0.22 2.70 ± 0.20 3.48 ± 0.61 1.91 ± 0.13 2.36 ± 0.10

PKcs [7–9,25]. Asp has been described as an activator of PKc in the case of enzymes purified from B. napus cell cultures [7], the banana fruit [8] and R. communis leaves [25] and seed [9]. A particular aspect of Asp mode of action is that it mainly functions by partially relieving the inhibitory effect of Glu [7,25]. Such coordinate regulation of PKc by amino acids renders respiratory metabolism particularly sensitive to metabolic feedback during nitrogen assimilation [1]. The fact that (6xHis)StPKc1 appears not regulated by amino acids could reflect the low importance of nitrogen metabolism in the control of PKc activity in the potato tuber. However, further support of this hypothesis should be sought by investigating the native form of the enzyme. Screening of effectors (Table 2) revealed that several intermediates of the TCA cycle (citrate, isocitrate, a-ketoglutarate and succinate) had an inhibitory effect on (6xHis)StPKc1 activity. The effect of pH on inhibition by these organic acids was not very strong. Among the tested metabolites, citrate was the most potent inhibitor, followed by a-ketoglutarate. These compounds were thus selected for further characterization. The effect of citrate and a-ketoglutarate was evaluated on PEP saturation kinetics at pHs 6.5, 6.8 and 7.5. No significant effect was observed on Vmax for any of the two inhibitors (not shown). However, the S0.5 value was progressively increased by raising concentrations of a-ketoglutarate between 0 and 25 mM and citrate between 0 and 10 mM (Table 3). The data indicated that citrate had more effect than a-ketoglutarate on PEP binding (e.g. compare S0.5 values obtained at 10 mM inhibitor concentration). The effect of varying pH between 6.5 and 7.5 on inhibition of (6xHis)StPKc1 by a-ketoglutarate was negligible. For example, the ratio of S0.5 values between pHs 6.5 and 7.5 was 1.72 in absence of effector and 1.79 in the presence of 25 mM inhibitor (Table 3). In the case of citrate, the same ratio was 2.13 in the presence of 10 mM inhibitor, indicating that decreasing pH leads to more inhibition of the enzyme by citrate. Overall these data indicate that citrate and a-ketoglutarate inhibited (6xHis)StPKc1 activity by reducing its affinity for PEP and that this effect was slightly more important under the acidic pH conditions that are thought to prevail in the hypoxic environment of the tuber. Previous reports have documented the inhibitory effect of citrate on native PKcs [7–9,25]. However, the inhibitory role of a-ketoglutarate appears to be less well characterized [7,25]. Our

In this study, we describe the conditions to express high levels of (6xHis)StPKc1 in E. coli in soluble form with the use of the GroES-GroEL chaperone complex. The recombinant protein was purified to electrophoretic homogeneity when an ATP buffer wash step was included in the purification protocol. (6xHis)StPKc1 had a high specific PK activity. The enzyme displayed characteristics of other PKcs such as heat stability and acidic pH optimum. The activity of (6xHis)StPKc1 was also found to be insensitive to regulation by amino acids whereas intermediates of the TCA cycle such as citrate acted as inhibitors. The advances made with this study will be useful to further our understanding of the regulation of this important enzyme. Acknowledgments This research was supported by a Discovery Grant from the National Science and Engineering Research Council of Canada (NSERC, RGPIN 227271) to Jean Rivoal. Evgenia L. Auslender acknowledges the support of an NSERC Undergraduate Student Research Award. Sébastien Dumont received a graduate fellowship from the Fonds Québécois de Recherche Nature et Technologie from the government of Québec. We wish to thank Drs. W.C. Plaxton and D.T. Dennis (Queen’s University) for the anti-B. napus PKc antibodies and the PKc cDNA, respectively. The help of Dr. Barry Shelp (University of Guelph) during the course of this study is also gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pep.2014.12.015. References [1] W.C. Plaxton, F.E. Podestà, The functional organization and control of plant respiration, Crit. Rev. Plant Sci. 25 (2006) 159–198. [2] C. Andre, J.E. Froehlich, M.R. Moll, C. Benning, A heteromeric plastidic pyruvate kinase complex involved in seed oil biosynthesis in Arabidopsis, Plant Cell 19 (2007) 2006–2022. [3] S.N. Oliver, J.E. Lunn, E. Urbanczyk-Wochniak, A. Lytovchenko, J.T. van Dongen, B. Faix, E. Schmalzlin, A.R. Fernie, P. Geigenberger, Decreased expression of cytosolic pyruvate kinase in potato tubers leads to a decline in pyruvate resulting in an in vivo repression of the alternative oxidase, Plant Physiol. 148 (2008) 1640–1654. [4] V.L. Knowles, S.G. McHugh, Z. Hu, D.T. Dennis, B.L. Miki, W.C. Plaxton, Altered growth of transgenic tobacco lacking leaf cytosolic pyruvate kinase, Plant Physiol. 116 (1998) 45–51. [5] B. Grodzinski, J. Jiao, V.L. Knowles, W.C. Plaxton, Photosynthesis and carbon partitioning in transgenic tobacco plants deficient in leaf cytosolic pyruvate kinase, Plant Physiol. 120 (1999) 887–896.

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