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J. Biol. Chem., Vol. 282, Issue 9, 6053-6060, March 2, 2007
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From the Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, Rosario, Argentina
Received for publication, October 5, 2006 , and in revised form, December 6, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In plants, both plastidic and cytosolic NADP-ME (EC 1.1.1.40 [EC] ) playing photosynthetic as well as non-photosynthetic roles have been identified (3). The photosynthetic plastidic isoform, which is expressed in bundle sheath cells of some C4 plants, represents a unique and specialized form of NADP-ME with particular kinetic, structural, and regulatory properties, and all of these make this enzyme suitable to participate in the CO2 concentrating mechanism that increases the photosynthetic yield of NADP-ME C4 plants (3, 4). In contrast, C4 non-photosynthetic NADP-ME isoforms are expressed in the cytosol (5) and plastids (6–9). Because these non-photosynthetic isoforms represent ancestors of the C4-specific enzyme they are ideal candidates for studying the evolution process toward the current C4-NADP-ME.
In maize, the plastidic non-photosynthetic NADP-ME (ZmChlMe2) represents the more recent and direct ancestor of the C4-NADP-ME (ZmChlMe1, Ref. 9). This enzyme is constitutively expressed in maize and plays housekeeping roles; in contrast to the C4 isoenzyme (8, 9). By cloning and expressing both the maize plastidic photosynthetic (C4) and non-photosynthetic NADP-ME (also known as non-C4-NADP-ME) as functional proteins in Escherichia coli, we identified several structural and kinetic differences between these two proteins (8, 10). Among these structural differences, the most relevant is the oligomeric state: the C4-NADP-ME assembles as a tetramer and the non-C4-NADP-ME as a dimer. With regard to the kinetic differences, one of the most outstanding is the inhibition of the C4-NADP-ME by the substrate malate in a pH-dependent way. The non-C4-NADP-ME is not inhibited by high concentrations of this substrate at all. This inhibition may be relevant in regulating the C4 isoform in vivo, by allowing the enzyme to increase its activity when photosynthesis is in progress.
The goal of the present work was to identify the segments of the primary structure of the C4 and non-C4 isoforms that are responsible for the C4-specific properties in maize NADP-ME. The high sequence similarity between these two enzymes (85%) allowed us to swap the corresponding segments between the two isoforms, constructing several chimeras. The structural and kinetic properties of the resulting chimerical enzymes were studied identifying segments involved in the tetramerization process and in the inhibition by the substrate malate at low pH.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Expression and Purification of Recombinant Chimerical NADP-ME—The pET-chimeric ME expression vectors contained a His tag sequence and the resulting fusion proteins were purified using a His-Bind column (Novagen). Induction and purification of the fusion proteins were done as previously described for the photosynthetic NADP-ME (10) and non-photosynthetic NADP-ME (8). Fusion enzymes were then concentrated on Centricon YM-30 (Amicon) and desalted using Buffer B (100 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 0,1 mM EDTA, 20% (v/v) glycerol, and 20 mM 
-mercaptoethanol). Purified fusion NADP-ME proteins were then incubated with enterokinase (1:100, w/w) in buffer B at 10 °C for 2 h to remove the N terminus encoded by the expression vector. Proteins were further purified by affinity chromatography (Affi-Gel Blue), and eluted with buffer C (100 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 500 mM NaCl, 0,1 mM EDTA, 20% (v/v) glycerol, and 20 mM -mercaptoethanol). Eluted enzymes were then concentrated and desalted on Centricon YM-30 (Amicon) using Buffer B. Purified enzymes were stored at –80 °C in Buffer B (with 50% glycerol) for further studies.
Circular Dichroism (CD) Measurements—CD spectra were obtained on a Jasco J-810 spectropolarimeter using a 1.0-cm path length cell and averaging five repetitive scans between 250 and 200 nm. Typically, 50 µgofC4, non-C4, or chimerical NADP-MEs in phosphate buffer (20 mM NaPi, pH 8.0, 5 mM MgCl2) were used for each assay.
NADP-ME Activity Assays—NADP-ME activity was determined spectrophotometrically as previously described (10). Initial velocity studies were performed by varying the concentration of one of the substrates around its Km while keeping the concentration of the other substrates at saturating levels. All kinetic parameters were calculated at least by triplicate determinations and subjected to non-linear regression. All substrate concentrations reported refer to the free, uncomplexed, reactant concentrations and were calculated considering the dissociation constant of the different complexes formed (Mg2+-NADP or Mg2+-malate). When testing different compounds as possible inhibitors or activators of the enzymatic activity, NADP-ME activity was measured in the presence of 2 or 5 mM of each effector (fumarate, succinate, citrate, 
-ketoglutarate, glucose-6-P, fructose 1,6-bisphosphate, or fructose-6-P), and saturating or non-saturating concentrations of malate (typically, 1/5 or 1/10 of the Km, malate value). When analyzing the inhibition of NADP-ME by high malate concentration at pH 7.0, the model that fitted best the kinetic data was
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x k1, the following expression for the initial rate of malate oxidative decarboxylation obtained,
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is the ratio between k2 and k1. This last value () should be less than 1, as the activity when the allosteric site is occupied is less than when it is empty. When no inhibition by high substrate concentration was found, the kinetic data obtained were fit to typical Michaelis-Menten equations. Gel Electrophoresis—SDS-PAGE was performed in 8 or 10% (w/v) polyacrylamide gels according to Laemmli (11). Proteins were visualized with Coomassie Blue or electroblotted onto a nitrocellulose membrane for immunoblotting. The membrane was incubated with affinity purified anti-maize recombinant photosynthetic NADP-ME antibodies (8). Bound antibodies were located with alkaline phosphatase-conjugated goat anti-rabbit IgG according to the manufacturer's instructions (Sigma).
Native PAGE was performed employing a 6% (w/v) polyacrylamide separating gel. Gels were analyzed by Western blotting or assayed for malic enzyme activity as described in Ref. 8.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-ketoglutarate, probably due to competition with the substrate malate. Other metabolites tested (glucose-6-P, fructose 1,6-bisphosphate, and fructose-6-P) did not show any significant effect on NADP-ME activity for either of the two isoforms. In this way, the pH-dependent inhibition of C4-NADP-ME by malate represents the major metabolite regulation difference found between maize photosynthetic and non-photosynthetic NADP-ME isoforms. Construction and Expression of Chimerical Maize NADP-ME—To examine the sequence determinants responsible for the structural and kinetic differences between both maize NADP-MEs, we constructed six chimeras of these proteins by interchanging different sequence segments using two conserved restriction sites (BamHI at position 306 and EcoRI at position 749 of the cDNA of maize C4-NADP-ME) (Fig. 1). We will refer to the C4 and non-C4-NADP-ME as L (leaf) and R (root), respectively, considering the tissues where the isoforms are highly expressed. The chimerical proteins were named: LR-NADP-ME, LpR-NADP-ME, RL-NADP-ME, RpL-NADP-ME, LRL-NADP-ME, and RLR-NADP-ME (Fig. 1). All of these chimerical proteins were successfully expressed in E. coli, and purified to homogeneity. They presented molecular masses between 62 and 66 kDa determined by SDS-PAGE and reacted with antibodies against maize recombinant C4-NADP-ME (not shown). They also presented NADP-ME activity, so they were kinetically and structurally characterized in the same way as the parental enzymes.
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Kinetic Characterization of Chimerical NADP-MEs at pH 8.0—Kinetic characterization of chimerical NADP-MEs, L-NADP-ME and R-NADP-ME was performed at pH 8.0 (optimum for the L and R isoforms, Table 1) and pH 7.0 (required for malate inhibition of the C4 isoform, Table 2).
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On the other hand, the Kmalate value at pH 8.0 for all the NADP-ME isoforms analyzed was very similar, with the L isoform showing the lower value followed by RpL-NADP-ME (Table 1). No inhibition by high malate concentration (up to 30 mM) was observed for any of the NADP-ME isoforms at pH 8.0 (Fig. 2).
Kinetic Characterization of Chimerical NADP-MEs at pH 7.0—Purified NADP-ME from maize green leaves is inhibited by high concentrations of the substrate malate in a pH-dependent way, that is, the inhibition is observed only at pH lower than 8.0 (12). This kinetic behavior was also observed when maize photosynthetic NADP-ME was expressed as a recombinant protein in E. coli (10), but was not observed in the case of maize recombinant non-photosynthetic NADP-ME (8). To locate the differences in the primary structure of these two enzymes that could account for malate inhibition, the activity of the chimerical NADP-MEs as a function of malate concentration was studied (Fig. 2 and Table 2).
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Malic enzyme activity as a function of malate concentration at pH 7.0 was assessed for the chimerical NADP-ME LR, LpR, RL, RpL, LRL, and RLR (Fig. 2C) to identify segments in the primary structure implicated in malate inhibition. RL-NADP-ME, RpL-NADP-ME, and LRL-NADP-ME were the only chimerical NADP-MEs analyzed showing inhibition by malate concentrations higher than 0.5 mM at pH 7.0 (Fig. 2C). Data obtained fitted very well to the same equation used in the case of the L isoform at pH 7.0 and the kinetic parameters obtained after data fitting were compared with the values obtained for the C4 isoform (Table 2). On the other hand, LR-NADP-ME, LpR-NADP-ME, and RLR-NADP-ME did not show inhibition by high malate concentrations at pH 7.0 (as the R isoform), and the Michaelis-Menten equation was the best-fitting model for the data obtained for these chimeras (Fig. 2C).
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With regard to the inhibition by high malate concentrations, the Kr,malate is very similar among the four NADP-MEs that showed inhibition (L isoform, RL-NADP-ME, RpL-NADP-ME, and LRL-NADP-ME). The decrease ratio in the kcat value () is also similar among these isoforms when malate is bound to the enzyme (Table 2).
Quaternary Structure of Chimerical NADP-ME from Maize—Previous work in our laboratory showed that recombinant L and R isoforms aggregate as tetramers or dimers, respectively (8, 10). The native molecular masses of both proteins were measured by size-exclusion chromatography and correlated with data obtained by native electrophoresis (8). Subsequently and to determine the factors associated with NADP-ME oligomerization, native activity gels were performed with the chimerical NADP-MEs obtained in the present work.
The data obtained indicate that whereas active forms of LR-NADP-ME and RpL-NADP-ME migrate in native electrophoresis as the L isoform (molecular mass consistent with a tetramer); RL-NADP-ME and LpR-NADP-ME migrate as the R isoform (molecular mass consistent with a dimeric state) (Fig. 3). These results indicate that the region located between amino acid residues 102 and 247 of the L isoform (flanked by BamHI and EcoRI restriction sites) is strongly associated with the oligomerization state of maize NADP-ME. In other words those chimerical proteins containing region 102–247 from the L isoform are tetrameric, whereas those carrying the homologous region from the R isoform are dimeric (Fig. 1).
To assess this hypothesis, the 145-residue region between BamHI and EcoRI restriction sites was exchanged between the L and R isoforms, obtaining the chimeras LRL-NADP-ME and RLR-NADP-ME. Native electrophoresis of these two proteins indicate that whereas the active form of LRL-NADP-ME migrate as a dimer, the chimera RLR-NADP-ME present an equilibrium between tetrameric and dimeric states (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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All the chimerical NADP-MEs obtained in the present work were functional. Nevertheless, the kcat obtained for LR-NADP-ME at both pH 7.0 and 8.0 was significantly lower than the value obtained for the other chimerical NADP-MEs, including the reciprocal RL-NADP-ME (Tables 1 and 2). However, all the Km values estimated for LR-NADP-ME are in the same range as those of others chimeras (Tables 1 and 2), suggesting that one or more residues are not appropriately located for catalysis in this particular enzyme, although the binding of substrates is not seriously affected.
The Allosteric Binding Site for Fumarate and Malate as Activators of Non-plant MEs Are Not Found in Maize NADP-ME—Human mitochondrial NAD(P)-ME can be regulated (activated) allosterically by fumarate, which binds at the dimer interface of this protein (14). By site-directed mutagenesis, 4 amino acid residues have been suggested to be involved in binding fumarate at this allosteric site: Arg-67, Arg-91, Glu-59, and Asp-102 (14, 16) (Fig. 4A). With regard to maize C4-NADP-ME, among the 4 residues found to be involved in fumarate activation of human NAD(P)-ME, only Asp-102 is conserved, which is in agreement with the fact that this enzyme is not activated by fumarate at all (Fig. 4A).
On the other hand, Ascaris suum NAD-ME is activated by both malate and fumarate, which bind to different binding sites that act synergically (17). Site-directed mutagenesis studies indicated that 2 amino acid residues were found to be implicated in this activation: Arg-105 (homologous to R91 of human NAD(P)-ME) and Lys-143 (Fig. 4A) (17). The effect of malate on A. suum NAD-ME is opposite to the effect of this substrate on C4-NADP-ME (Fig. 2) and, in correlation with this, the residue Arg-105 involved in malate activation in A. suum NAD-ME (17) is not conserved in C4-NADP-ME (Fig. 4A). On the other hand, these residues present homologues in non-C4-NADP-ME (Fig. 4A), which is not activated nor inhibited by malate at all, suggesting that other amino acid residues are necessary for activation of ME by malate.
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When considering substrate inhibition, three general mechanisms appear (13): pseudo-substrate inhibition, where the substrate affects some assay component rather than the enzyme directly; kinetic substrate inhibition, in which the substrate interferes or alters the catalytic mechanism; and allosteric substrate inhibition, in which the substrate binds a site different from the active site causing inhibition by a conformational change. In the present work, when assaying malate inhibition, free metal concentration was maintained constant, and therefore this could not be the cause of malate inhibition. Moreover, inhibition studies were performed in identical conditions for all isoforms tested, ruling out the possibility of a pseudo-inhibition mechanism (Fig. 2). In this way, the inhibition by high malate concentration of maize photosynthetic NADP-ME could be due to a kinetic substrate inhibition or, alternatively, to an allosteric malate binding site. Malate inhibition at pH 7.0 but not at pH 8.0 was observed for four NADP-ME isoforms: L-NADP-ME, RL-NADP-ME, RpL-NADP-ME, and LRL-NADP-ME (Fig. 2). These proteins share a common region that extends from residue 249 to the C terminus of the C4-NADP-ME isoform, suggesting that this sequence is associated with the inhibition mechanism (Fig. 1). The fact that inhibition by high malate concentration was observed only at pH 7.0 and was associated to a limited region of the protein, prompted us to consider the hypothesis of an allosteric site responsible for such inhibition. In correlation with this, the kinetic data obtained for the NADP-ME isoforms that were inhibited (Fig. 2) fitted very well an equation that considers an allosteric inhibitor binding site for malate.
The lack of inhibition at pH 8.0 could be due to the impossibility of malate to bind to the proposed allosteric site at this pH. In this case, the residues involved in this process should be different between C4 and non-C4-NADP-ME, as should be their protonation state between pH 7.0 and 8.0, which in turn would determine the differential kinetic behavior under both conditions. Taking into account these statements, several amino acid residues are good candidates to be involved in this inhibition (not shown), and their role remains to be determined by mutation studies.
Pigeon liver NADP-ME was also found to be inhibited by high concentrations of malate, inhibition that decreased when increasing the metal cofactor concentration (18, 24). This inhibition was initially explained by a model of either pre-existing non-identical malate binding sites in the enzyme tetramer or by negative cooperative between initially identical binding sites, as different methods indicated the presence of heterogeneous malate sites in the enzyme (18, 19). Nevertheless, crystal structure of this enzyme does not provide evidence of asymmetry among the four subunits (15), so further studies are needed to characterize the molecular basis for the reactivity of half of the sites of pigeon liver malic enzyme.
The Mechanism of Oligomerization of Maize NADP-ME Differs Markedly from Other Malic Enzymes—By analyzing the maize NADP-ME chimeras constructed in the present work, we identified a region between amino acid residues 102 and 247 of the C4 isoform and its homologue region in the non-C4 isoform, which is strongly associated with the particular active oligomeric state of each maize NADP-ME (Figs. 1 and 4B). It is interesting to note that the isoform LRL-NADP-ME, which contained the mentioned region from the non-C4 isoform (Fig. 3), does not tetramerize, indicating that some residues from this region are essential for tetramerization. On the other hand, the isoform RLR-NADP-ME exhibits an equilibrium between tetrameric and dimeric states (Fig. 3), indicating that the intermediate segment originated from the C4 isoform induces tetramerization, but not in a complete way. The isoforms LR-NADP-ME and RpL-NADP-ME can completely tetramerize (Fig. 3), indicating that other interactions of the mentioned region contribute also to the oligomerization process but are not strong enough to drive complete tetramerization by their own. In this way, it seems that, for complete tetramerization, the region between amino acid residues 102 and 247 is indispensable, along with some residues from the N or, alternatively, C terminus of the L-NADP-ME. Although this oligomerization-related segment presents a high degree of conservation between these two NADP-ME isoforms, several non-conservative amino acid residue substitutions can be identified (Fig. 4B) that could be responsible, at least partially, for the different oligomeric states of these isoforms. The fact that the plastidic A. thaliana isoform presents similar homology to both the tetrameric and dimeric maize MEs in the region postulated to be involved in tetramerization (Fig. 4B), and that it also has an intermediate oligomeric state (equilibrium between tetramer and dimer, Ref. 29) reinforces the conclusion that this region has an essential role in tetramerization. On the other hand, the functional significance of tetrameric or dimeric states is not obvious from the present work, as both dimeric and tetrameric chimerical isoforms presented, in several cases, almost the same catalytic efficiency and substrate affinities.
With regard to the tetramer interaction of human NAD(P)-ME, it has been determined that it is mediated by a segment of 
20 residues at the C terminus of the enzyme monomer (1). Nevertheless, a multiple segment alignment of maize NADP-ME isoforms, and the NAD(P)-MEs for which crystal structures are available (Fig. 4C), clearly shows that this part of the protein is not present in maize NADP-ME nor in other plant-sequenced NADP-MEs (not shown). In this way, plant NADP-MEs have a different strategy for tetramerization and different amino acidic residues involved in the tetramer interface. Moreover, it has been shown that mutation of Trp-548 of pigeon liver NADP-ME, which is completely buried at the tetramer interface, has a tremendous effect in the quaternary structure of the enzyme (23). This residue, which is conserved among crystallized NAD(P)-MEs, is absent from maize NADP-ME (Fig. 4C). Besides this particular residue, the region between residues 541 and 544 of human NAD(P)-ME also contributes to the formation of the tetramer interface (1). Specifically, mutation of Arg-542 of human NAD(P)-ME at the tetrameric interfacial exo site that binds ATP, resulted in dimeric mutants (25). Nevertheless, this residue is conserved in both dimeric and tetrameric maize isoforms (Fig. 4C). These observations indicate that the oligomerization interactions of plant NADP-MEs clearly differ from those of animal NADP-MEs.
Evolution of C4-NADP-ME from a Non-C4 Isoform: Regulation by Malate of Key C4 Isoforms—The results obtained here give new insights into the special requirements of C4-NADP-ME, in relation to C3 isoforms, to be well suitable for C4 photosynthesis. In this regard, C4-NADP-ME presents notably more affinity for the substrates, nearly 10 times lower KNADP and 2 times lower Kmalate, than the C3 enzyme (Table 1). However, the molecular determinants for the higher substrate affinity of the C4-NADP-ME could not be mapped by the approach used in the present work, as the affinity for the substrates of all the chimerical NADP-ME analyzed was between the values obtained for the parent enzymes (Table 1). Thus, the molecular determinants involved in the higher substrate affinity of the C4-NADP-ME may be distributed in several segments of the protein.
On the contrary, the other relevant kinetic difference, the inhibition by malate in a pH-dependent way, could be mapped associated to the C terminus of the C4-NADP-ME. In this regard, the C4-NADP-ME from Flaveria bidentis (20) and sugarcane (21) are also inhibited by high malate concentrations. Although more C4-NADP-ME family members should be analyzed, it is probable that this inhibition may be important for the C4-pathway regulation in vivo. In this way, the high level of malate concentration found in plant tissues (22) and the decrease in pH observed in chloroplasts in darkness, would both produce a decrease in NADP-ME activity when carbon fixation is not active in C4 plants.
Another C4 enzyme that evolved from C3 counterparts that were analyzed is P-enolpyruvate carboxylase (PEPC) (26, 27). The C4 and C3 PEPCs presented, as the case of NADP-ME, nearly the same kcat, but differed drastically in the affinity for PEP, which was opposite to NADP-ME, lower for the C4 isoform. An increase in the inhibition constant for malate is also observed in the C4 isoform. With regards to regulation, the C4 PEPC activity is regulated by diurnal phosphorylation, which decreases the degree of inhibition by malate of the C4 enzyme. In this way, in the case of PEPC, the phosphorylation/dephosphorylation of the enzyme is involved in the activation/inhibition of the enzyme by malate. This was also the case for the PEPC from C4 single cell-type photosynthesis (28).
In summary, it seems that during the evolution to C4 enzymes, apart from a higher level and selective expression (PEPC in mesophyll and NADP-ME in bundle sheath cells), changes in kinetic and regulatory properties were also necessary to adapt the enzymes to new metabolic contexts. It is not surprising that, in both C4 PEPC and NADP-ME evolution, the inhibition of the high enzyme activity found in C4 leaves is controlled by the same metabolite (malate), which mediates activity inhibition when C4 photosynthesis is off.
Concluding Remarks—In the present work, we demonstrate that maize NADP-ME and crystallized non-plant NAD(P)-MEs present markedly different mechanisms for oligomerization and allosteric regulation. The active site of these enzymes present significant conservation, whereas their corresponding regulatory mechanism and oligomerization interactions are more divergent. These observations suggest that the emergence of different types of allosteric sites during the evolution of NAD(P)-MEs, led to the creation of enzymes with unique regulatory mechanisms and best suited to fulfill different metabolic roles.
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FOOTNOTES |
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1 Fellows of the Consejo Nacional de Investigaciones Científicas y Técnicas.
2 Members of the Researcher Career of the Consejo Nacional de Investigaciones Científicas y Técnicas.
3 To whom correspondence should be addressed. Tel.: 54-341-4371955; Fax: 54-341-4370044; E-mail: candreo{at}fbioyf.unr.edu.ar.
4 The abbreviations used are: NADP-ME, NAD(P)-malic enzyme; PEPC, phospho-enolpyruvate carboxylase.
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