Identification of domains involved in the allosteric regulation of cytosolic Arabidopsis thaliana NADP-malic enzymes Mariel C. Gerrard Wheeler 1 , Cintia L. Arias 1 , Vero ´ nica G. Maurino 2 , Carlos S. Andreo 1 and Marı ´ a F. Drincovich 1 1 Centro de Estudios Fotosinte ´ ticos y Bioquı ´ micos, Universidad Nacional de Rosario, Argentina 2 Botanisches Institut, Universita ¨ tzuKo ¨ ln, Cologne, Germany Introduction Malic enzymes (MEs) catalyse the reversible oxidative decarboxylation of l-malate to pyruvate, CO 2 and NAD(P)H in the presence of a divalent cation [1]. This enzyme is widely distributed in nature, as the substrates and products of the reaction participate in different met- abolic pathways. In plants, photosynthetic and nonpho- tosynthetic NADP-dependent isoenzymes (NADP-ME; EC 1.1.1.40) have been found in both plastids and cytosol [2]. Moreover, the co-expression of different NADP-ME isoenzymes has been observed in the same cell, and even in the same subcellular compartment [3]. The elucidation of the biological role of the different NADP-ME isoenzymes, apart from being involved in C 4 photosynthesis or crassulacean acid metabolism, will Keywords allosteric; Arabidopsis thaliana; isoenzymes; NADP-malic enzyme; regulation Correspondence M. F. Drincovich, Centro de Estudios Fotosinte ´ ticos y Bioquı ´ micos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina Fax: 54 341 4370044 Tel: 54 341 4371955 E-mail: drincovich@cefobi-conicet.gov.ar (Received 19 June 2009, revised 28 July 2009, accepted 4 August 2009) doi:10.1111/j.1742-4658.2009.07258.x The Arabidopsis thaliana genome contains four genes encoding NADP- malic enzymes (NADP-ME1–4). Two isoenzymes, NADP-ME2 and NADP-ME3, which are shown to be located in the cytosol, share a remarkably high degree of identity (90%). However, they display different expression patterns and show distinct kinetic properties, especially with regard to their regulation by effectors, in both the forward (malate oxida- tive decarboxylation) and reverse (pyruvate reductive carboxylation) reac- tions. In order to identify the domains in the primary structure that could be responsible for the regulatory differences, four chimeras between these isoenzymes were constructed and analysed. All chimeric versions exhibited the same native structures as the parental proteins. Analysis of the chime- ras constructed indicated that the region from amino acid residue 303 to the C-terminal end of NADP-ME2 is critical for fumarate activation. However, the region flanked by amino acid residues 303 and 500 of NADP-ME3 is involved in the pH-dependent inhibition by high malate concentration. Furthermore, the N-terminal region of NADP-ME2 is necessary for the activation by succinate of the reverse reaction. Overall, the results show that NADP-ME2 and NADP-ME3 are able to distinguish and interact differently with similar C 4 acids as a result of minimal struc- tural differences. Therefore, although the active sites of NADP-ME2 and NADP-ME3 are highly conserved, both isoenzymes acquire different allo- steric sites, leading to the creation of proteins with unique regulatory mech- anisms, probably best suited to the specific organ and developmental pattern of expression of each isoenzyme. Abbreviations GFP, green fluorescent protein; ME, malic enzyme. FEBS Journal 276 (2009) 5665–5677 ª 2009 The Authors Journal compilation ª 2009 FEBS 5665 require further effort, as the gene family of this protein is more complex than expected [4]. The Arabidopsis thaliana genome contains four NADP-ME genes [3,4]. One gene encodes a plastidic enzyme (NADP-ME4 [3]), but the other three isoenzymes do not possess predictable organellar sorting sequences and thus are thought to be located in the cytosol (NADP-ME1–3). Previous studies have indicated dif- ferential expression patterns for each isoenzyme [3]. In this regard, although NADP-ME2 and NADP- ME4 are constitutively expressed in mature organs, NADP-ME1 is restricted to secondary roots and NADP-ME3 to trichomes and pollen [3]. Although the four isoenzymes share a high degree of identity (75–90%), the recombinant enzymes show distinct structural and kinetic properties [3,5]. Specifically, the isoenzymes behave differently in terms of regula- tion by metabolic effectors, NADP-ME2 being the most highly regulated, especially by activation [5]. In particular, NADP-ME2 and NADP-ME3 share 90% identity (Fig. 1), are encoded in the same chromosome and belong to the cytosolic dicot group in a phylogenetic tree constructed with plant NADP- ME sequences [3]. In the malate oxidative decar- boxylation reaction, although NADP-ME2 is highly activated by aspartate, fumarate and succinate, NADP-ME3 is inhibited by fumarate with no modifi- cation of the enzymatic activity in the presence of aspartate and succinate [5]. Furthermore, although suc- cinate and fumarate show strong activation of the NADP-ME2 pyruvate reductive decarboxylation reac- tion (up to 400%), these metabolites act as inhibitors of the NADP-ME3 reverse reaction [5]. Two NADP- ME2 amino-terminal deletions previously analysed indicated that some residues from this region are criti- cal for aspartate and CoA activation [5]. However, regions involved in the differential regulation by fuma- rate and succinate could not be mapped by this approach. Moreover, the mutation of R115 in NADP- ME2 indicated that this amino acid residue is involved in fumarate activation [5]. However, this residue is conserved in NADP-ME3, indicating that other amino Fig. 1. Sequence alignment of A. thaliana NADP-ME2 and NADP-ME3. Regions of the primary structure of each isoenzyme that are involved in fumarate activation (in yellow), CoA activation (underlined) and malate inhibition (in green) of the forward reaction are highlighted. In addition, the regions involved in succinate activation of the reverse reaction are highlighted in light blue. Nonconserved residues between the two sequences are shown in bold. ‘*’, iden- tical residues; ‘:’, conserved substitution; ‘.’, semiconserved substitution. Regulation of A. thaliana NADP-malic enzyme activity M. C. Gerrard Wheeler et al. 5666 FEBS Journal 276 (2009) 5665–5677 ª 2009 The Authors Journal compilation ª 2009 FEBS acid residues are responsible for the differential regula- tion by fumarate. In this work, NADP-ME2 (Uniprot Accession Number Q9LYG3) and NADP-ME3 (Uniprot Accession Number Q9XGZ0) are experimen- tally shown to be located in the cytosol; moreover, the relationship between the primary structure and differ- ences in regulation was investigated by the character- ization of complementary chimeras between the two isoenzymes. The segments swapped in the construction of the chimeras allowed us to evaluate the eight non- conserved amino acid residues between NADP-ME2 and NADP-ME3. These amino acid residues are sepa- rated into two regions: three are located in the first segment swapped and five in the second (Fig. 1). Using this approach, specific segments of the primary struc- ture responsible for regulatory differences were identi- fied, indicating that minimal structural changes are responsible for the distinct behaviour of these two highly similar NADP-ME isoenzymes. Results Subcellular localization of A. thaliana NADP-ME2 and NADP-ME3 In order to determine the subcellular localization of A. thaliana NADP-ME2 and NADP-ME3, the full- length cDNA of each isoenzyme was fused in frame to the green fluorescent protein (GFP) coding sequence, and the localization of the fluorescence was assayed by transient expression in A. thaliana cell cultures. Figure 2 clearly shows that NADP-ME2 and NADP-ME3 are both homogeneously distributed in the cytosol. A con- trol assay with the GFP coding region shows the locali- zation of free GFP in the cytosol and nucleus (Fig. 2). Structural characterization of chimeric NADP-MEs In order to examine the sequence domains responsible for regulatory differences between the cytosolic isoen- zymes NADP-ME2 and NADP-ME3, four chimeric proteins (named ME2.3, ME2.3¢, ME3.2 and ME3.2¢; Fig. 3) were successfully expressed in E. coli and puri- fied to homogeneity. To determine whether the chime- ric proteins display any structural changes in relation to the parental proteins, CD spectra for all chimeric and parental enzymes were compared. In all cases, the CD spectra obtained after corrections for protein con- centration were very similar (data not shown), indicat- ing that there was no significant loss of secondary structure in the chimeric proteins. Monomeric molecular masses of 65 kDa were determined by SDS-PAGE for all chimeras (data not Fig. 2. Subcellular localization of A. thaliana NADP-ME2 and NADP-ME3. Transient expression of 35S::NADP-ME2::GFP, 35S::NADP- ME3::GFP and 35S::GFP in A. thaliana protoplasts. Bright field images with the superimposed GFP fluorescence images shown at the top. Fluorescence distribution is shown at the bottom. The scale bar represents 12 lm. M. C. Gerrard Wheeler et al. Regulation of A. thaliana NADP-malic enzyme activity FEBS Journal 276 (2009) 5665–5677 ª 2009 The Authors Journal compilation ª 2009 FEBS 5667 shown), which are in accordance with those obtained for the parental recombinant proteins [3]. Native electrophoresis of the purified proteins indi- cated that the parental and chimeric proteins presented almost identical electrophoretic mobility (data not shown and [3]). Moreover, the parental and chimeric proteins presented highly similar native molecular masses by gel filtration chromatography, Fig. 3. Chimeric NADP-MEs constructed and analysed in the present work. The conserved restriction sites EcoRV and BclI at positions 910 and 1500, respectively, of the cDNA of parental enzymes (NADP-ME2 and NADP-ME3) were used to construct the complementary chimeric enzymes ME2.3, ME2.3¢, ME3.2 and ME3.2¢. These sites correspond to positions 303 and 500, respectively, in the protein sequence of NADP-ME2 and NADP-ME3. The recombinant NADP-MEs that are activated by fumarate or CoA or inhibited by high malate concentration at pH 7.0 (for the forward reaction) or that are activated by succinate (for the reverse reaction) are indicated on the right by ‘4’. Regions of the primary structure of each parental NADP-ME that are involved in fumarate, CoA and succinate activation and malate inhibition are indicated. Table 1. Properties of parental and chimeric NADP-MEs. The indicated values are the average of at least three different measure- ments ± SD. For k cat calculations, a 65 kDa monomeric molecular mass was used for all isoenzymes. Some values for parental NADP-ME2 and NADP-ME3, obtained previously [3], are included for comparison. (k catD ⁄ k catC , k cat decarboxylation ⁄ k cat carboxylation ; M, monomeric molecular mass; N, native molecular mass; NI, no inhibition was observed.). NADP-ME2 NADP-ME3 ME2.3 ME2.3¢ ME3.2 ME3.2¢ Malate oxidative decarboxylation, pH 7.5 k cat (s )1 ) 324 ± 29 268 ± 24 148 ± 13 156 ± 16 45 ± 5 94 ± 13 c K mNADP (lM) 72 ± 7 6.5 ± 0.6 31 ± 5 55 ± 9 23 ± 1 31 ± 8 c K m L -malate (mM) 3.3 ± 0.4 0.8 ± 0.1 1.1 ± 0.1 4.8 ± 0.5 2.3 ± 0.4 1.7 ± 0.1 c Malate oxidative decarboxylation; pH 7.0 K r (mM) NI 0.6 ± 0.1 9.6 ± 0.2 NI NI 1.1 ± 0.1 F NI 0.1 ± 0.05 0.7 ± 0.1 NI NI 0.3 ± 0.1 Pyruvate reductive carboxylation; pH 7.0 k cat (s )1 ) 75 ± 3 237 ± 11 164 ± 16 19 ± 2 27 ± 3 181 ± 19 K mpyruvate (mM) 0.5 ± 0.05 5.0 ± 0.2 7.9 ± 0.3 2.1 ± 0.2 2.4 ± 0.3 31 ± 4.2 Relation forward ⁄ reverse reaction k catD ⁄ k catC 4.3 1.1 0.9 8.3 1.7 0.5 Structural properties M (kDa) a 65 65 65 65 65 65 N (kDa) b 243 249 228 255 234 241 a Determined by SDS-PAGE. b Determined by gel filtration chromatography. c At pH 8.0, as inhibition by high malate concentration was observed at values lower than pH 8.0. Regulation of A. thaliana NADP-malic enzyme activity M. C. Gerrard Wheeler et al. 5668 FEBS Journal 276 (2009) 5665–5677 ª 2009 The Authors Journal compilation ª 2009 FEBS which were consistent with tetrameric oligomeric states (Table 1). Kinetic characterization of chimeric NADP-MEs in the oxidative decarboxylation direction Previous results have indicated that NADP-ME2 dis- plays higher decarboxylation activity at a lower pH (optimum pH 6.8) than NADP-ME3 (optimum pH 7.7) [5]. Nevertheless, as at pH 7.5 both isoenzymes retained 95% of the maximal activity (data not shown), this pH was chosen for the comparative char- acterization of the kinetic and regulatory properties of the chimeric proteins (Table 1). As ME3.2¢ was inhib- ited by high malate concentration at pH values lower than pH 8.0 (data not shown), the kinetic analysis of this chimera was performed at pH 8.0 (Table 1). All chimeras showed less specific activity than the parental isoenzymes (Table 1). The k cat values obtained were between 48% and 14% of the value obtained for NADP-ME2, the parental enzyme with the highest specific activity (Table 1). The K mNADP and K ml-malate values of the chimeric proteins were of the same order of magnitude as those of NADP-ME2 (Table 1). These results indicate that, despite the differences detected, the binding sites for the substrates were integral in the chimeric proteins. Regulatory properties of the chimeric NADP-MEs in the oxidative decarboxylation direction Several compounds were tested as possible effectors of the enzymatic activity of each chimeric NADP-ME in the direction of the oxidative decarboxylation of l-malate (Fig. 4), and compared with the results obtained with the parental isoenzymes [5]. With the exception of acetyl-CoA and CoA, the effectors were tested at two concentrations, 0.5 and 2 mm, which are referred to as low and high concentrations, respectively. As in the case of NADP-ME2 and NADP-ME3, oxaloacetate and ATP were the strongest inhibitors of the enzymatic activity of all chimeric proteins (Fig. 4). ME2.3¢ and ME3.2 were inhibited only by high ATP concentration, whereas all other chimeras were inhib- ited by both high and low ATP concentrations, proba- bly because of the higher affinity for this inhibitor. Glucose-6-phosphate also inhibited the enzymatic activity of all proteins (Fig. 4), although oxaloacetate and ATP were the strongest inhibitors. The effects of acetyl-CoA and CoA on the enzy- matic activity were tested at 20 lm (Fig. 4). Acetyl- CoA did not modify significantly the activity of the chimeras. However, CoA maintained its status as an activator in the chimeric proteins ME2.3¢ and ME3.2, whereas no modification of ME2.3 and ME3.2¢ activi- ties were observed in the presence of this compound (Fig. 4). Furthermore, both succinate and aspartate were able to activate all chimeric enzymes, although activation by succinate was observed only at high concentration in the case of ME2.3 and ME3.2¢ (Fig. 4). By contrast, fumarate activated only ME3.2 and inhibited ME3.2¢, but only at high concentration in the latter case (Fig. 4). Kinetic characterization of chimeric NADP-MEs in the oxidative decarboxylation direction at pH 7.0 The effect of the substrate l-malate on the forward reaction of NADP-ME2 and NADP-ME3 was analy- sed at pH 7.0. The kinetic measurements showed that NADP-ME3 was partially inhibited by high concentra- tions of l-malate at this pH (Fig. 5). The kinetic data obtained for NADP-ME3 at pH 7.0 fitted to an equa- tion in which two different sites for malate, one cata- lytic and one allosteric, are considered (see Materials and methods [6]). When occupied, the allosteric site decreases the activity of the enzyme, rendering a par- tial inhibition that is characterized by a K r value of 0.6 and u value of 0.1 (Table 1). The inhibition of NADP- ME3 by high malate concentration was pH dependent, as no inhibition was observed at pH 7.5 (Table 1 and [3]). However, NADP-ME2 was not inhibited by high malate concentration at pH 7.0 (Fig. 5). In order to identify sequence segments in the pri- mary structure of NADP-ME2 and NADP-ME3 responsible for the differential behaviour at pH 7.0, the chimeric proteins were analysed at this pH. The results indicated that only ME2.3 and ME3.2¢ were inhibited by high malate concentration. The data obtained for these enzymes at pH 7.0 fitted well to the equation that considers that the enzyme binds malate at two different sites, one catalytic and the other allo- steric, as in the case of NADP-ME3 (data not shown). The K r values obtained (Table 1) indicate that the malate allosteric site of ME2.3 and ME3.2¢ displays lower affinity than that of the parental enzyme NADP-ME3. In turn, the higher u parameters for the chimeras indicate a smaller decrease in the catalytic activity when the allosteric site is occupied by the inhibitor, in comparison with the parental enzyme NADP-ME3 (Table 1). In the case of ME3.2 ¢, the inhi- bition by high malate concentration was also observed at pH 7.5 (not shown), but not at pH 8.0, which was used for the kinetic characterization of the chimera (Table 1). M. C. Gerrard Wheeler et al. Regulation of A. thaliana NADP-malic enzyme activity FEBS Journal 276 (2009) 5665–5677 ª 2009 The Authors Journal compilation ª 2009 FEBS 5669 Reversibility of the reaction catalysed by chimeric NADP-MEs The four chimeric NADP-MEs were tested for their capability to catalyse the reverse reaction: the pyru- vate reductive carboxylation. As the carboxylation reaction catalysed by the parental isoenzymes showed an optimum at pH 7.0 [5], this pH value was used for kinetic analysis of the chimeras. All chimeric pro- teins showed less specific activity than NADP-ME3 (Table 1). ME2.3 and ME3.2¢ are the chimeras with the highest k cat values for the reverse reaction, with Fig. 4. Regulatory properties of the chimeric NADP-ME isoenzymes in the oxidative decarboxylation direction. NADP-ME forward activity was measured for each isoenzyme at pH 7.5 in the absence or presence of 0.5 or 2 m M of each effector [indicated as Succinate 0.5 or 2; Fumarate 0.5 or 2; Asp (aspartate) 0.5 or 2; OAA (oxaloacetate) 0.5 or 2; ATP 0.5 or 2; Glucose 6P (glucose-6-phosphate) 0.5 or 2] or 20 l M of CoA or acetyl-CoA. The results are presented as the percentage of activity in the presence of the effectors in relation to the activity mea- sured in the absence of the metabolites for each of the respective enzyme constructs. The assays were performed at least in triplicate and the error bars indicate SD. Significant inhibition (as indicated in Materials and methods): dark grey and single-hatched bars. Significant activa- tion (as indicated in Materials and methods): light grey and double-hatched bars. The results for parental NADP-ME2 and NADP-ME3, obtained previously [5], are included for comparison. Regulation of A. thaliana NADP-malic enzyme activity M. C. Gerrard Wheeler et al. 5670 FEBS Journal 276 (2009) 5665–5677 ª 2009 The Authors Journal compilation ª 2009 FEBS values even higher than that of NADP-ME2 (Table 1). These proteins were able to catalyse the reductive carboxylation reaction at higher rates than the oxidative decarboxylation reaction (Table 1). However, ME2.3 and ME3.2¢ display the lowest affin- ity towards pyruvate in comparison with all the other isoenzymes (Table 1). Regulatory properties of the chimeric NADP-MEs in the reductive carboxylation direction The effect of several metabolites on the reductive carboxylation reaction of the chimeric proteins was analysed and compared with the results obtained with the parental enzymes (Fig. 6). l-Malate, one of the products of the reverse reac- tion, was the strongest inhibitor of the enzymatic activ- ity of all the chimeric versions (Fig. 6). Aspartate also inhibited the reductive carboxylation of all chimeras and NADP-ME2, but did not modify the enzymatic activity of NADP-ME3 (Fig. 6). With regard to succinate, this organic acid activated the chimeric enzymes possessing the amino-terminal region of NADP-ME2, ME2.3 and ME2.3¢ (Fig. 6). By contrast, succinate did not modify the activity of ME3.2 and ME3.2¢ (Fig. 6). In the case of fumarate, all chimeras showed activation by this compound (Fig. 6), as did NADP-ME2, whereas the parental iso- enzyme NADP-ME3 was inhibited by both succinate and fumarate [5]. Discussion A. thaliana NADP-ME2 and NADP-ME3 are shown to be located in the cytosol. The measurement of enzymatic activity in the presence of several putative metabolic effectors indicated distinct regulatory pat- terns for both isoenzymes (Figs 4–6). In order to iden- tify the key sequence regions associated with the more relevant kinetic differences between these highly similar isoenzymes, several chimeras of these proteins were constructed and analysed. All the chimeric proteins showed structural integrity by CD analysis and conser- vation of the quaternary conformation (Table 1). Thus, the absence of severe structural changes with respect to the parental enzymes, and the fact that the chimeras were functional proteins (Table 1), allowed us to use them as a tool to compare regulatory pat- terns and to evaluate the determinants of the primary sequence associated with them. Regulatory regions associated with fumarate and CoA activation of the malate oxidative decarboxylation reaction of NADP-ME2 Several compounds were tested as possible modifiers of the malate oxidative decarboxylation reaction cataly- sed by NADP-ME2 and NADP-ME3. Oxaloacetate, ATP, glucose-6-phosphate and acetyl-CoA similarly affected the activity of both native enzymes and chime- ras (Fig. 4 [5]). However, succinate, fumarate, aspar- tate and CoA produced differential effects on the activity of NADP-ME2 and NADP-ME3, as well as the different chimeras analysed (Fig. 4). Fumarate activation was observed for the parental NADP-ME2 and the chimeric enzyme ME3.2 (Fig. 4). These proteins share a common region, which extends from amino acid residue 303 to the C-terminal end of NADP-ME2, suggesting that this sequence is associ- ated with the activation mechanism by this compound (Figs 1 and 3). Moreover, amino acid residues from both segments swapped (from amino acid residue 303 Fig. 5. NADP-ME2 and NADP-ME3 forward activity as a function of malate concentration at pH 7.0. Free Mg 2+ and NADP concentrations were kept constant at 10 and 1.0 m M, respectively, in all cases. A typical result is shown from at least three independent determinations. The data were fitted to the Michaelis–Menten equation for NADP-ME2 or to the model described in Materials and methods for NADP-ME3 (see equation in [6]), and are presented as the percentage of maximum activity. The absolute values corresponding to 100% of activity are 497 and 218 UÆmg )1 for NADP-ME2 and NADP-ME3, respectively. Malate inhibition was not observed for either isoenzyme at pH 7.5 (Table 1 and [3]). M. C. Gerrard Wheeler et al. Regulation of A. thaliana NADP-malic enzyme activity FEBS Journal 276 (2009) 5665–5677 ª 2009 The Authors Journal compilation ª 2009 FEBS 5671 to 500 and from amino acid residue 500 to the car- boxyl-terminal end of NADP-ME2; Figs 1 and 3) are involved in this regulation, as the chimeras ME2.3¢ and ME 3.2¢, both bearing only one of these segments, are not activated by fumarate (Fig. 3). Like NADP-ME2, human mitochondrial NAD(P)- ME is allosterically activated by fumarate [7]. In this case, fumarate binds at the dimer interface, where four amino acid residues are involved: R67, R91, E59 and D102 [7–9]. Only two of these amino acid Fig. 6. Regulatory properties of the chimeric NADP-ME isoenzymes in the reductive carboxylation direction. NADP-ME reverse activity was measured for each isoenzyme at pH 7.0 in the absence or presence of 1, 7.5 or 15 m M of each effector [indicated as L-malate, Succinate, Fumarate and Asp (aspartate) 1, 7.5 and 15 m M]. The results are presented as the percentage of activity in the presence of the effectors in relation to the activity measured in the absence of the metabolites, for each of the respective enzyme constructs. The assays were performed at least in triplicate, and error bars indicate SD. Significant inhibition (as indicated in Materials and methods): dark grey and single- hatched bars. Significant activation (as indicated in Materials and methods): light grey and double-hatched bars. The results for parental NADP-ME2 and NADP-ME3, obtained previously [5], are included for comparison. Regulation of A. thaliana NADP-malic enzyme activity M. C. Gerrard Wheeler et al. 5672 FEBS Journal 276 (2009) 5665–5677 ª 2009 The Authors Journal compilation ª 2009 FEBS residues (R91 and D102; homologous to R115 and D126 of NADP-ME2, respectively) are conserved in A. thaliana NADP-ME2 (Fig. 1 and [5]), suggesting that the mechanism of activation should be different between the two isoenzymes. Moreover, these two amino acid residues are also conserved in NADP- ME3 (Fig. 1), which is not activated by fumarate (Fig. 4). Therefore, other amino acid residues differ- ent from those proposed for the human isoenzyme [7–9] are necessary to control the binding capacity and fumarate response of NADP-ME2. Several amino acid residues in this C-terminal region (Figs 1 and 3) are good candidates to be involved in this activation, and their role remains to be determined by mutational studies. Specifically, from the five non- conserved amino acid residues in the suggested domain involved in fumarate activation, the muta- tions at positions 357 and⁄ or 360 could be involved in the differential regulation, as well as the conserved change at position 543 (Fig. 1). The activation of NADP-ME2 by fumarate could be relevant in vivo,asA. thaliana accumulates large amounts of fumarate and malate during the day and uses these organic acids as a way to transport carbon to other organs, and as energy and carbon sources in conditions of energy demand [10,11]. In this sense, the activation by fumarate of NADP-ME2, which is expressed in photosynthetic and nonphotosynthetic organs of A. thaliana, may be linked to the higher utilization of organic acids on energy demand by the activation of this isoenzyme when the fumarate con- centration increases. However, our data suggest that NADP-ME3, which is restricted to pollen and tri- chomes, is not linked to this organic acid utilization and regulation. By contrast, the region between amino acid residues 303 and 500 of NADP-ME2 may be associated with CoA activation of the l-malate decarboxylation reac- tion because only NADP-ME2, ME2.3¢and ME3.2 showed activation by this compound (Figs 1 and 3). Previous studies have indicated that the deletion of 44 amino acid residues from the amino-terminal region of NADP-ME2 provides an enzyme that is not activated at all by CoA [5]. Thus, the activation by this metabo- lite may require the participation of the region flanked by amino acid residues 303 and 500 of NADP-ME2 interacting with residues from the amino-terminal region. Although NADP-ME3 is not activated by aspar- tate and succinate (Fig. 4), it is surprising that the activity of the four chimeras increases in the pres- ence of both effectors, although to a different extent (Fig. 4). In this way, it can be inferred that the acti- vation by these metabolites is mediated by several amino acid residues, not found in NADP-ME3, but distributed in the different protein segments of NADP-ME2 that were swapped by the construction of the chimeras. Regulatory region associated with the pH-dependent malate inhibition of NADP-ME3 Malate inhibition of the forward reaction at pH 7.0 was observed only in the case of NADP-ME3, ME2.3 and ME3.2¢ (Fig. 3, Table 1). These proteins share a common region, which extends from amino acid resi- dues 303 to 500 of NADP-ME3, suggesting that this sequence is associated with the mechanism of substrate inhibition (Figs 1 and 3). The fact that the inhibition by high substrate concentration was associated with a limited region of the protein supports the hypothesis of the existence of an allosteric site responsible for such regulation, as in the case of maize photosynthetic NADP-ME [6]. In agreement with this, the kinetic data obtained for the enzymes that were inhibited by l-malate fitted very well to the equation that considers an allosteric inhibitor binding site for malate (Fig. 4 [6]). Moreover, as the inhibition by malate is pH dependent (Table 1), it is concluded that the amino acid residue(s) involved in this allosteric regulation may change the protonation state between pH 7.0 and pH 7.5, leading to the loss of inhibition at higher pH. In the particular case of ME3.2¢, the loss of malate inhibition is observed at higher pH (pH 8.0, Table 1). It is thus possible that in this chimera a change in the pKa value of the amino acid residue(s) involved in malate inhibition may occur as a result of interaction with different amino acid residues in the allosteric site. The amino acid residue changes at positions 357, 420 and ⁄ or 481 between NADP-ME2 and NADP-ME3 are good candidates for involvement in the pH-dependent regulation by malate, as they involve changes from noncharged amino acid residues in NADP-ME2 to positive amino acid residues, depending on pH, in NADP-ME3 (Fig. 1). The inhibition by excess l-malate was marked as a pH-dependent characteristic of MEs implicated in C 4 photosynthesis [6]. This in vivo regulatory mechanism was suggested to occur through the pH change induced in illuminated chloroplasts, and ensures that NADP- ME is fully active only at pH 8.0, when carbon fixa- tion is in progress. Thus, the pH-dependent malate inhibition of NADP-ME3 was unexpected, as it is a cytosolic isoenzyme not implicated in photosynthesis. In this way, this isoenzyme from a C 3 species displays the feature of malate inhibition at pH 7.0 associated M. C. Gerrard Wheeler et al. Regulation of A. thaliana NADP-malic enzyme activity FEBS Journal 276 (2009) 5665–5677 ª 2009 The Authors Journal compilation ª 2009 FEBS 5673 with C 4 photosynthesis, probably as an evolutionary ancestor of C 4 NADP-ME. Similarly, a nonphoto- synthetic recombinant NADP-ME from tobacco also showed partial inhibition by l-malate [12]. Further studies should be conducted to reveal whether the pH-dependent regulation of a nonphotosynthetic isoenzyme may be relevant in vivo, especially with regard to the localization of NADP-ME3 in the cyto- sol of pollen and trichome cells. Regulatory region associated with succinate activation of the pyruvate reductive carboxylation reaction of NADP-ME2 The activation of the pyruvate reductive carboxylation reaction by succinate was only observed in the case of NADP-ME2 and the chimeras ME2.3 and ME2.3¢ (Fig. 6). Thus, a regulatory region associated with acti- vation by this metabolite could be defined, which com- prises the first 303 amino-terminal amino acid residues of NADP-ME2 (Figs 1 and 3). However, the three nonconserved amino acid changes between NADP- ME2 and NADP-ME3 are located in the amino-termi- nal region (Fig. 1), which is not involved in succinate activation, as NADP-ME2 lacking the first 44 amino acid residues is still activated by succinate [5]. Thus, some of the semiconserved or conserved amino acid residue changes may be involved in succinate activa- tion. Good candidates are the changes in charge at positions 253 and ⁄ or 295 (Fig. 1). By contrast, fumarate was able to activate the reverse reaction catalysed by NADP-ME2 (Fig. 6 [5]). However, although NADP-ME3 is not activated by this compound at all, it is surprising that the activity of the four chimeras is increased by fumarate (Fig. 6). In this regard, ME3.2¢, the chimera that shares the minimum number of amino acid residues with NADP- ME2, is less activated by this compound than the other compounds (Fig. 6). In this way, amino acid res- idues of different segments of NADP-ME2 swapped in the construction of the chimeras are involved in the fumarate activation of the pyruvate reductive carboxyl- ation reaction. However, the different degree of fuma- rate activation shown by the chimeras (e.g. 150% in the case of ME3.2 and 671% in the case of ME2.3¢ with 7.5 mm of fumarate) may indicate that some regions are more critical than others in the activation by this allosteric modulator. This hypothesis should be tested by site-directed mutagenesis of candidate amino acid residues from the different regions and by estima- tion of the kinetic parameters of fumarate activation. Aspartate inhibits NADP-ME2 and all the chimeras analysed, but is unable to decrease NADP-ME3 activ- ity, although tested at high concentration (Fig. 6). These results are consistent with an allosteric type of inhibition, in which amino acid residues from the dif- ferent segments used to construct the chimeras are involved. However, as some chimeras are inhibited only at high aspartate concentrations (e.g. ME3.2¢ is not inhibited at all at 1 mm aspartate), some segments of the primary structure seem to be more critical than others (Fig. 6). This hypothesis can be tested by site- directed mutagenesis of candidate amino acid residues from the different regions, and by estimation of the affinity towards aspartate as inhibitor. The forward and reverse reactions are distinctly regulated by effectors which are associated with different protein determinants Finally, the results obtained suggest that the regulation of the forward and reverse NADP-ME activities is mediated by different protein regions (Figs 4 and 6). In this sense, the dual effect of aspartate in the NADP-ME2 reaction, which activates the decarboxyl- ation and inhibits the carboxylation reaction, can be explained through a conformational change in the enzyme induced by the substrate malate, which can be important for exposing an aspartate-activating binding site [5]. By contrast, succinate and fumarate strongly increased the activity of NADP-ME2 in both direc- tions of the reaction (Figs 4 and 6). However, despite the structural similarity between these two organic acids, the kinetic results indicated that the activation by these compounds was mediated by different bind- ing sites. This conclusion is supported by previous studies, which showed that R115 mutation of NADP- ME2 abolished the activating effect of fumarate, but did not modify the activity measured in the presence of succinate [5]. In turn, our experimental data clearly indicate that, for each separate metabolite, the regula- tion of the direct reaction is mediated by different sites than in the reverse reaction (Fig. 3). Again, conformational changes induced in the protein by l-malate or pyruvate could provide an explanation for these observations. A future challenge will be to determine the three-dimensional structure of a plant NADP-ME in the presence and absence of the sub- strates to analyse the conformational changes that are induced by the binding of the substrate, which may influence the observed allosteric regulation of each isoenzyme. As suggested recently, a knowledge of the allosteric interactions could be very useful in protein design inhibition or activation to influence protein function as required [13]. Regulation of A. thaliana NADP-malic enzyme activity M. C. Gerrard Wheeler et al. 5674 FEBS Journal 276 (2009) 5665–5677 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... all involved in the modulation of NADP-ME activity Nevertheless, although these compounds are structurally similar, NADP-ME2 and NADP-ME3 are able to distinguish and interact differently with these C4 acids as a result of minimal differences in the primary protein structure (Fig 1) The precise identification of these differences will be important for the enzymatic engineering of NADP-ME aimed at creating... Wheeler et al Concluding remarks Although A thaliana NADP-ME2 and NADP-ME3 share a high identity, they display distinct regulatory properties Thus, the activities of these two isoenzymes, which colocalize in the cytosol, are modulated by different effectors In this work, the regions involved in some of these differential regulations were mapped in the primary structure of each isoenzyme Succinate, fumarate,... was measured at pH 7.0 in the absence or presence of 1.0, 7.5 or 15 mm l-malate, succinate, fumarate or aspartate, keeping the pyruvate concentration at the Km value for each isoenzyme (Table 1) The results are presented as the percentage of activity in the presence of the effector in relation to the activity measured in the absence of the FEBS Journal 276 (2009) 5665–5677 ª 2009 The Authors Journal compilation... carboxylation of pyruvate (reverse reaction) was measured in an assay medium containing 50 mm Mops ⁄ KOH pH 7.0, 10 mm MgCl2, 0.2 mm NADPH, 10 mm NaHCO3 and 50 mm pyruvate in a final volume of 0.5 mL The linearity of the reaction was monitored to detect any CO2 loss during the assay One unit (U) is defined as the amount of enzyme that catalyses the formation or consumption of 1 lmol of NAD(P)H per minute under the. .. activity measurements in the presence of a given metabolite were significantly different from the activity measured in its absence (P < 0.05) was activation or inhibition indicated as significant in Figs 4 and 6 Moreover, inhibitions lower than 80% and activations higher than 140% relative to the activity in the absence of the metabolites are indicated When analysing the inhibition of NADP-ME by high malate... treated with the corresponding restriction endonucleases, and the fragments obtained were purified and recombined to obtain chimeric sequences in the expression vector pET32 The plasmids constructed were named as follows: pET-ME2.3, pET-ME2.3¢, pET-ME3.2 Regulation of A thaliana NADP-malic enzyme activity and pET-ME3.2¢ (Fig 3) The chimeric constructs were sequenced to verify the correct swapping of the fragments... protein by a nickel-containing HisBind column (Novagen, Gibbstown, New York, USA) The induction and isolation of the proteins were performed as described previously [14,15] The fusion proteins were digested with enterokinase as described in [3], and the proteins were further purified using an affinity Affi Gel Blue column (BioRad, Hercules, CA, USA) and analysed by SDS-PAGE The purified enzymes were concentrated... uncritical: does Arabidopsis need six malic enzyme isoforms? Plant Sci 176, 715–721 Gerrard Wheeler MC, Arias CL, Tronconi MA, Maurino VG, Andreo CS & Drincovich MF (2008) Arabidopsis thaliana NADP-malic enzyme isoforms: high degree of identity but clearly distinct properties Plant Mol Biol 67, 231–242 ´ Detarsio E, Alvarez CE, Saigo M, Andreo CS & Drincovich MF (2007) Identification of domains involved in tetramerization... mm)1Æcm)1) Initial velocity studies were performed by varying the concentration of one of the substrates around its Km value, whilst keeping the other substrate concentrations at saturating levels All kinetic parameters were calculated at least by triplicate determinations, and adjusted to nonlinear regression using free concentrations of all substrates [14] When testing different compounds as possible inhibitors... creating enzymes able to fulfil desired reactions responding to specific effectors Materials and methods Heterologous expression and purification of the recombinant enzymes The pET32 vectors containing the full-length cDNA sequences of NADP-ME2 and NADP-ME3, pET-ME2 and pET-ME3 [3], were used to express each NADP-ME fused in- frame to a histidine tag to facilitate the purification of the expressed fusion protein . Identification of domains involved in the allosteric regulation of cytosolic Arabidopsis thaliana NADP-malic enzymes Mariel C. Gerrard Wheeler 1 , Cintia. as the percentage of activity in the presence of the effectors in relation to the activity measured in the absence of the metabolites, for each of the