Identification of Domains Involved in Tetramerization and Malate Inhibition of Maize C4-NADP-Malic Enzyme*

C4 photosynthetic NADP-malic enzyme (ME) has evolved from non-C4 isoforms and gained unique kinetic and structural properties during this process. To identify the domains responsible for the structural and kinetic differences between maize C4 and non-C4-NADP-ME several chimeras between these isoforms were constructed and analyzed. By using this approach, we found that the region flanked by amino acid residues 102 and 247 is critical for the tetrameric state of C4-NADP-ME. In this way, the oligomerization strategy of these NADP-ME isoforms differs markedly from the one that present non-plant NADP-ME with known crystal structures. On the other hand, the region from residue 248 to the C-terminal end of the C4 isoform is involved in the inhibition by high malate concentrations at pH 7.0. The inhibition pattern of the C4-NADP-ME and some of the chimeras suggested an allosteric site responsible for such behavior. This pH-dependent inhibition could be important for regulation of the C4 isoform in vivo, with the enzyme presenting maximum activity while photosynthesis is in progress.

C 4 photosynthetic NADP-malic enzyme (ME) has evolved from non-C 4 isoforms and gained unique kinetic and structural properties during this process. To identify the domains responsible for the structural and kinetic differences between maize C 4 and non-C 4 -NADP-ME several chimeras between these isoforms were constructed and analyzed. By using this approach, we found that the region flanked by amino acid residues 102 and 247 is critical for the tetrameric state of C 4 -NADP-ME. In this way, the oligomerization strategy of these NADP-ME isoforms differs markedly from the one that present non-plant NADP-ME with known crystal structures. On the other hand, the region from residue 248 to the C-terminal end of the C 4 isoform is involved in the inhibition by high malate concentrations at pH 7.0. The inhibition pattern of the C 4 -NADP-ME and some of the chimeras suggested an allosteric site responsible for such behavior. This pH-dependent inhibition could be important for regulation of the C 4 isoform in vivo, with the enzyme presenting maximum activity while photosynthesis is in progress.
NAD(P)-malic enzyme (NAD(P)-ME) 4 (EC 1.1.1.38, 1.1.1.39, and 1.1.1.40) catalyzes the reversible oxidative decarboxylation of L-malate to yield carbon dioxide and pyruvate with the concomitant reduction of NAD(P) (1). The enzyme is found in most living organisms, because the products of the reaction are used as a source of carbon and reductive power in different cell compartments. The amino acid sequences of malic enzymes are highly conserved among many organisms, from bacteria to human. However, malic enzymes do not show recognizable amino acid sequence homology to other proteins such as dehydrogenases, decarboxylases, or other oxidative decarboxylases, comprising a new class of oxidative decarboxylases (2).
In plants, both plastidic and cytosolic NADP-ME (EC 1.1.1.40) playing photosynthetic as well as non-photosynthetic roles have been identified (3). The photosynthetic plastidic iso-form, which is expressed in bundle sheath cells of some C 4 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 CO 2 concentrating mechanism that increases the photosynthetic yield of NADP-ME C 4 plants (3,4). In contrast, C 4 nonphotosynthetic NADP-ME isoforms are expressed in the cytosol (5) and plastids (6 -9). Because these non-photosynthetic isoforms represent ancestors of the C 4 -specific enzyme they are ideal candidates for studying the evolution process toward the current C 4 -NADP-ME.
In maize, the plastidic non-photosynthetic NADP-ME (ZmChlMe2) represents the more recent and direct ancestor of the C 4 -NADP-ME (ZmChlMe1, Ref. 9). This enzyme is constitutively expressed in maize and plays housekeeping roles; in contrast to the C 4 isoenzyme (8,9). By cloning and expressing both the maize plastidic photosynthetic (C 4 ) and non-photosynthetic NADP-ME (also known as non-C 4 -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 C 4 -NADP-ME assembles as a tetramer and the non-C 4 -NADP-ME as a dimer. With regard to the kinetic differences, one of the most outstanding is the inhibition of the C 4 -NADP-ME by the substrate malate in a pH-dependent way. The non-C 4 -NADP-ME is not inhibited by high concentrations of this substrate at all. This inhibition may be relevant in regulating the C 4 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 C 4 and non-C 4 isoforms that are responsible for the C 4 -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.

EXPERIMENTAL PROCEDURES
Construction of Chimerical NADP-ME Sequences-Maize C 4 (pET-ME, Ref. 10) and non-C 4 -NADP-ME (pET-RME, Ref. 8) sequences were inserted into the pET32 expression vector (Novagen). The sequences correspond to the mature proteins (without transit peptides) in both cases. The plasmids pET-ME and pET-RME were treated with the corresponding restriction * This work was supported by Consejo Nacional de Investigaciones Científicas y Té cnicas PIP number 3029, Agencia Nacional de Promoció n Científica y Tecnoló gica PICT number 01-11604, and Fundació n Antorchas Project N°4248-63. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Fellows of the Consejo Nacional de Investigaciones Científicas y Té cnicas. 2 Members of the Researcher Career of the Consejo Nacional de Investigaciones Científicas y Té cnicas. 3 To whom correspondence should be addressed. Tel.: 54-341-4371955; Fax: 54-341-4370044; E-mail: candreo@fbioyf.unr.edu.ar. 4 The abbreviations used are: NADP-ME, NAD(P)-malic enzyme; PEPC, phospho-enolpyruvate carboxylase. endonucleases ( Fig. 1) and the fragments obtained were purified and recombined to obtain the chimerical NADP-ME sequences into the expression vector pET-32. The plasmids obtained were named as follows: pET-LR-NADP-ME, pET-RL-NADP-ME, pET-RpL-NADP-ME, and pET-LpR-NADP-ME (see Fig. 1). Two more plasmids (pET-LRL-NADP-ME and pET-RLR-NADP-ME) were constructed by changing only the segment between the cleavage sites for the restriction enzymes BamHI and EcoRI (Fig. 1). The inserts of all the chimerical constructs were sequenced to verify correct swapping of the corresponding fragments and that no mistakes were made due to subcloning procedures. BL21(DE3) E. coli was then transformed with the pET plasmid containing the chimerical NADP-MEs. 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 MgCl 2 , 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 MgCl 2 , 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 Ϫ80°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 g of C 4 , non-C 4 , or chimerical NADP-MEs in phosphate buffer (20 mM NaP i , pH 8.0, 5 mM MgCl 2 ) 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 K m 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 (Mg 2ϩ -NADP or Mg 2ϩ -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 1,6bisphosphate, or fructose-6-P), and saturating or non-saturating concentrations of malate (typically, 1/5 or 1/10 of the K m , 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 in which the free enzyme is capable of binding malate at two different sites, one catalytic and the other allosteric (regulatory). In this kinetic scheme, E symbolizes the enzyme, M c the malate bound to the catalytic binding site (with dissociation constant K c ), M r malate bound to the regulatory allosteric site (with dissociation constant K r ), and P the products of the enzymatic reaction. Thus, two different catalytic constants are postulated, namely k 1 and k 2 , to indicate product formation when the allosteric site is empty or occupied, respectively. Considering k 2 ϭ ϫ k 1 , the following expression for the initial rate of malate oxidative decarboxylation obtained, where v is the activity measured at a specific malate concentration (M); V max is the maximum activity; K c and K r are the dissociation constants of the catalytic and regulatory site, respectively; and is the ratio between k 2 and k 1 . 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 antirabbit 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.

Metabolites Tested as Effectors of Maize C 4 and Non-C 4 -
NADP-ME-To detect other regulatory differences between maize C 4 and non-C 4 -NADP-ME, in addition to the previously reported malate inhibition at pH 7.0 of the C 4 isoform, we analyzed several metabolites as effectors of the activity of these two enzymes. No definitive activation by fumarate or succinate was found for both maize NADP-ME isoforms, although these metabolites were characterized as activators of other MEs. On the other hand, partial inhibition of both isoforms (up to 40%) was found in the case of citrate and ␣-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 C 4 -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 C 4 -NADP-ME) (Fig. 1). We will refer to the C 4 and non-C 4 -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 chi-merical 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 C 4 -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.
To determine whether the construction of the chimerical proteins resulted in a loss of overall structural integrity, CD spectra for all chimerical and parental proteins were compared. In all cases, the CD spectra obtained were superimposable after corrections for protein concentration (not shown). In this way, the structural changes should be minimal, without causing a severe loss of protein secondary structure.
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 C 4 isoform, Table 2).
The chimerical NADP-MEs analyzed (RL, LR, LpR, RpL, LRL, and RLR) showed less specific activity (k cat ) than the L and R isoforms at pH 8.0 ( Table 1). The k cat values obtained for RL, RpL, and LpR were between 39 and 58% of the value obtained for the L isoform; whereas LRL and RLR presented considerably lower specific activities, between 2 and 12% of the value for the L isoform (Table 1). However, chimerical protein LR-NADP-ME displayed the lowest k cat value of all the recombinant proteins analyzed (more than 2 orders of magnitude, Table 1). Nevertheless, the K NADP of this chimerical enzyme was more than 2 times lower than that for the non-C 4 counterpart (Table 1), indicating that, at least, the binding site for NADP is integral in this chimerical NADP-ME. The catalytic efficiency for the non-C 4 and chimerical NADP-MEs when using NADP as substrate ranged from nearly 1 to 3 orders of magnitude lower than for the C 4 counterpart (Table 1).
On the other hand, the K malate 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 FIGURE 1. Chimerical NADP-MEs obtained and analyzed in the present work. The conserved restriction sites BamHI and EcoRI of the parental enzymes (L, C 4 -NADP-ME; and R, non-C 4 -NADP-ME) were used to construct the reciprocal chimerical enzymes LR-NADP-ME (LR), LpR-NADP-ME (LpR), RL-NADP-ME (RL), RpL-NADP-ME (LpR), LRL-NADP (LRL), and RLR-NADP-ME (RLR) are indicated. Native oligomeric state (T, tetramer; or D, dimer) as well as inhibition by high malate concentrations at pH 7.0 (ϩ and Ϫ indicate inhibition or no inhibition, respectively) of each recombinant NADP-ME are indicated on the right. Regions implicated in the oligomerization state and malate inhibition are indicated at the bottom.  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). First of all, a detailed study of the activity of recombinant L and R isoforms as a function of malate concentration at both pH 7.0 or 8.0 was performed. Fig. 2 clearly shows that inhibition by malate concentrations higher than 0.5 mM is observed for the L isoform at pH 7.0 but not at pH 8.0 (Fig. 2). Data obtained for the L isoform (pH 8.0, L8) and the R isoform (pH 7.0 and 8.0; R7 and R8; Fig. 2) were fit to a typical Michaelis-Menten equation, yielding parameter values indicated in Tables 1 and 2. On the other hand, in the case of the L isoform at pH 7.0 (L7, Fig. 2), the data obtained fitted very well to an equation in which two different binding sites for malate are considered, one catalytic (K c,malate ) and the other allosteric (K r,malate ) (see Model 1 and Equation 1 under "Experimental Procedures"). This second allosteric site decreases the activity of the enzyme when occupied, rendering a partial inhibition of the activity. It is worth mentioning that lower regression fitting values were obtained when kinetic data were fit to other models and equations for inhibition by high substrate concentrations (13).
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 C 4 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 . NADP-ME activity as a function of malate concentration at pH 7.0 or 8.0. A and B, C 4 -NADP-ME (L isoform) and non-C 4 -NADP-ME (R isoform) activity as a function of malate concentration at pH 7.0 (L7 and R7) and pH 8.0 (L8 and R8). For better viewing, two separate graphs for low malate concentrations (A) and high malate concentrations (B) are presented. C and D, percentage of maximum NADP-ME activity as a function of free malate concentration for C 4 -NADP-ME (L7 and L8), non-C 4 -NADP-ME (R7 and R8), LR-NADP-ME (LR7 and LR8), LpR-NADP-ME (LpR7 and LpR8), RL-NADP-ME (RL7 and RL8), RpL-NADP-ME (RpL7 and RpL8), LRL-NADP-ME (LRL7 and LRL8), and RLR-NADP-ME (RLR7 and RLR8) at pH 7.0 (C) or 8.0 (D). For a clearer comparison of the malate inhibition effect, data are presented as a % of the maximum activity obtained for each enzyme. The k cat values of the isoforms are indicated in Tables 1 and 2 and the absolute values differ among the different enzymes as indicated there. Free Mg 2ϩ and NADP concentrations were kept constant at 10 and 0.5 mM, respectively, in all cases. A typical result is shown from at least four independent determinations, and data fitting to Michaelis-Menten or to the equation indicated under "Experimental Procedures" (when inhibition was observed) is shown in each case.

TABLE 2 Kinetic parameters assayed at pH 7.0 for the recombinant maize C 4 -NADP-ME (10), the non-C 4 -NADP-ME (8), and the chimerical NADP-MEs obtained in the present work
Values are given as average Ϯ S.D. Each value is the average for at least two different preparations of each enzyme. When inhibition by high malate concentration was observed, K c,malate and K r,malate values were obtained using the equation indicated under "Experimental Procedures." equation was the best-fitting model for the data obtained for these chimeras (Fig. 2C). Table 2 shows a summary of the kinetic parameters obtained at pH 7.0 for the chimerical NADP-MEs, in comparison to the L and R isoforms. When inhibition by high malate concentrations is observed, the data fitted correctly the equation indicated under "Experimental Procedures," obtaining two different binding constants for malate, one catalytic (K c,malate ) and the other regulatory (K r,malate ). When comparing the k cat , lower values are observed at pH 7.0 versus 8.0 for all the recombinant NADP-MEs, with the exception of RpL-NADP-ME and LRL-NADP-ME (Table 1 versus 2). Nevertheless, the decrease in k cat at pH 7.0 range from 47% (L isoform) to only 16%, as is the case for the R isoform. The K c,malate at pH 7.0 for the L isoform, RpL-NADP-ME, and LRL-NADP-ME is nearly 1 order of magnitude lower at pH 7.0 than at 8.0, and for the case of RL-NADP-ME, is nearly 3 times lower. However, in the case of the R isoform and all the other chimerical NADP-MEs that are not inhibited by malate, this value is almost invariant when comparing measurements at pH 7.0 versus 8.0. In this way, a correlation between lower K c,malate values at pH 7.0 than at pH 8.0 and inhibition by high malate concentration is observed (Table 2).

Parameters
With regard to the inhibition by high malate concentrations, the K r,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 k cat 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).

DISCUSSION
Because the C 4 -NADP-ME isoenzyme (L isoform) has evolved from the non-C 4 -NADP-ME (R isoform), we searched for changes in the primary structure of maize C 4 -NADP-ME responsible for its C 4 properties, allowing it to participate in a highly active metabolism such as photosynthesis. The results obtained analyzing different recombinant chimeras of the maize L and R isoforms allowed us to find key sequence elements associated with the more relevant kinetic and structural differences between these isoforms: inhibition by malate and oligomeric state.
All the chimerical NADP-MEs obtained in the present work were functional. Nevertheless, the k cat 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 K m 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 C 4 -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 C 4 -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 C 4 -NADP-ME (Fig. 4A). On the other hand, these residues present homologues in non-C 4 -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.
Maize C 4 -NADP-ME Presents a Regulatory Region Associated to Malate Inhibition-When analyzing maize C 4 -NADP-ME inhibition by malate, we first examined if the oligomeric state of this isoform (tetrameric) was associated to the inhibition by high malate concentration. This would have been the case if the malate binding site responsible for malate inhibition was located in the interface between the dimers, as is the case of the allosteric site for fumarate in the human NADP-ME (14), and, possibly, the allosteric activating site for malate in A. suum NAD-ME (17). Alternatively, if the inhibition was related to heterogeneity of the malate active binding sites in the tetramer, as was suggested for the pigeon liver NADP-ME (18,19) (see below), it was reasonable that the tetramer (C 4 -NADP-ME) was inhibited by malate, whereas the dimer (non-C 4 -NADP-ME) was not. Nevertheless, analysis of the chimera obtained in the present work indicates that no correlation is observed between the oligomeric state and malate inhibition at all (Fig. 1), so these two first working hypotheses were discarded. Thus, the mechanism of maize C 4 -NADP-ME inhibition by malate was not in accordance with other kinetic mechanisms previously reported for non-plant MEs.
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 FIGURE 4. A, allosteric fumarate binding site in crystallized non-plant MEs. Multiple sequence alignments were performed among maize C 4 and non-C 4 -NADP-ME, and malic enzymes with known crystal structure. Alignments were restricted to those areas identified as part of the fumarate (human NAD(P)-ME) and malate and fumarate (A. suum NAD-ME) allosteric binding sites. The amino acid residues underlined and highlighted have been identified by site-directed mutagenesis to be involved in activation by fumarate and malate (14,16,17). B, residues of the region involved in the oligomerization state of maize NADP-MEs. Multiple sequence alignments were performed among maize C 4 and non-C 4 -NADP-ME, and malic enzymes with known crystal structure. Alignments were restricted to the area postulated to be involved in tetramerization in maize NADP-MEs (Fig. 1). The Arabidopsis thaliana plastidic NADP-ME (NADP-ME4, Ref. 29) is included for comparison in this case. Residues involved in tetramerization interactions in the crystallized enzymes are underlined and highlighted (1). C, postulated amino acids contributing to the formation of the tetramer of crystallized non-plant MEs. Multiple sequence alignments were performed among maize C 4 and non-C 4 -NADP-ME, and malic enzymes with known crystal structure. Alignments are restricted to the C terminus region that is crucial for tetramerization in the crystallized enzymes. Residues involved in tetramerization interactions in the crystallized enzymes are underlined and highlighted (1).
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 C 4 -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 C 4 and non-C 4 -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 C 4 isoform and its homologue region in the non-C 4 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-C 4 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 C 4 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 ϳ20 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 C 4 -NADP-ME from a Non-C 4 Isoform: Regulation by Malate of Key C 4 Isoforms-The results obtained here give new insights into the special requirements of C 4 -NADP-ME, in relation to C 3 isoforms, to be well suitable for C 4 photosynthesis. In this regard, C 4 -NADP-ME presents notably more affinity for the substrates, nearly 10 times lower K NADP and 2 times lower K malate , than the C 3 enzyme (Table 1). However, the molecular determinants for the higher substrate affinity of the C 4 -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 C 4 -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 C 4 -NADP-ME. In this regard, the C 4 -NADP-ME from Flaveria bidentis (20) and sugarcane (21) are also inhibited by high malate concentrations. Although more C 4 -NADP-ME family members should be analyzed, it is probable that this inhibition may be important for the C 4 -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 C 4 plants.
Another C 4 enzyme that evolved from C 3 counterparts that were analyzed is P-enolpyruvate carboxylase (PEPC) (26,27). The C 4 and C 3 PEPCs presented, as the case of NADP-ME, nearly the same k cat , but differed drastically in the affinity for PEP, which was opposite to NADP-ME, lower for the C 4 isoform. An increase in the inhibition constant for malate is also observed in the C 4 isoform. With regards to regulation, the C 4 PEPC activity is regulated by diurnal phosphorylation, which decreases the degree of inhibition by malate of the C 4 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 C 4 single cell-type photosynthesis (28).
In summary, it seems that during the evolution to C 4 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 C 4 PEPC and NADP-ME evolution, the inhibition of the high enzyme activity found in C 4 leaves is controlled by the same metabolite (malate), which mediates activity inhibition when C 4 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 reg-ulatory mechanisms and best suited to fulfill different metabolic roles.