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.

Among the different isoforms of NADP-malic enzyme (NADP-ME) involved in a wide range of metabolic pathways in plants, the NADP-ME that participates in C(4)-photosynthesis is the most studied. In the present work, the expression in E. coli of a cDNA encoding for a maize non-photosynthetic NADP-ME is presented. The recombinant NADP-ME thus obtained presents kinetic and structural properties different from the enzyme previously purified from etiolated leaves and roots. Moreover, the recombinant non-photosynthetic NADP-ME presents very high intrinsic NADP-ME activity, which is unexpected for a non-C( 4) NADP-ME. Using antibodies against this recombinant enzyme, an immunoreactive band of 66 kDa is detected in different maize tissues indicating that the 66 kDa-NADP-ME is in fact a protein expressed in vivo. The recombinant NADP-ME assembles as a dimer, although the results obtained indicate that a higher molecular mass oligomeric state of the enzyme is found in maize roots in vivo. In this way, maize presents at least three NADP-ME isoforms: a 72 kDa constitutive form (previously characterized); the novel non-photosynthetic 66 kDa isoform characterized in this work (which is the product of the ZmChlMe2 gene and the likely precursor to the evolution of the photosynthetic C(4) NADP-ME) and the 62 kDa isoform (implicated in C(4) photosynthesis). The contribution of the present work anticipates further studies concerning the equilibrium between the oligomeric states of the NADP-ME isoforms and the evolution towards the C(4) isoenzyme in maize.

Two highly similar plastidic NADP-malic enzymes (NADP-MEs) are found in the C(4) species maize (Zea mays); one exclusively expressed in the bundle sheath cells (BSCs) and involved in C(4) photosynthesis (ZmC(4)-NADP-ME); and the other (ZmnonC(4)-NADP-ME) with housekeeping roles. In the present work, these two NADP-MEs were analyzed regarding their redox-dependent activity modulation. The results clearly show that ZmC(4)-NADP-ME is the only one modulated by redox status, and that its oxidation produces a conformational change limiting the catalytic process, although inducing higher affinity binding of the substrates. The reversal of ZmC(4)-NADP-ME oxidation by chemical reductants suggests the presence of thiol groups able to form disulfide bonds. In order to identify the cysteine residues involved in the activity modulation, site-directed mutagenesis and MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) analysis of ZmC(4)-NADP-ME were performed. The results obtained allowed the identification of Cys192, Cys246 (not conserved in ZmnonC(4)-NADP-ME), Cys270 and Cys410 as directly or indirectly implicated in ZmC(4)-NADP-ME redox modulation. These residues may be involved in forming disulfide bridge(s) or in the modulation of the oxidation of critical residues. Overall, the results indicate that, besides having acquired a high level of expression and localization in BSCs, ZmC(4)-NADP-ME displays a particular redox modulation, which may be required to accomplish the C(4) photosynthetic metabolism. Therefore, the present work could provide new insights into the regulatory mechanisms potentially involved in the recruitment of genes for the C(4) pathway during evolution.

Malic enzymes catalyze the oxidative decarboxylation of l-malate to yield pyruvate, CO(2), and NAD(P)H in the presence of a bivalent metal ion. In plants, different isoforms of the NADP-malic enzyme (NADP-ME) are involved in a wide range of metabolic pathways. The C(4)-specific NADP-ME has evolved from C(3)-type malic enzymes to represent a unique and specialized form of NADP-ME as indicated by its particular kinetic and regulatory properties. In the present study, the mature C(4)-specific NADP-ME of maize was expressed in Escherichia coli. The recombinant enzyme has essentially the same physicochemical properties and K(m) for the substrates as those of the naturally occurring NADP-ME previously characterized. However, the k(cat) was almost 7-fold higher, which may suggest that the previously purified enzyme from maize leaves was partially inactive. The recombinant NADP-ME also has a very low intrinsic NAD-dependent activity. Five mutants of NADP-ME at the postulated putative NADP-binding site(s) (Gsite5V, Gsite2V, A392G, A387G, and R237L) were constructed by site-directed mutagenesis and purified to homogeneity. The participation of these residues in substrate binding and/or the catalytic reaction was inferred by kinetic measurements and circular dichroism and intrinsic fluorescence spectra. The results obtained were compared with a predicted three-dimensional model of maize C(4) NADP-ME based on crystallographic studies of related animal NAD(P)-MEs. The data presented here represent the first prokaryotic expression of a plant NADP-ME and reveals valuable insight regarding the participation of the mutated amino acids in the binding of substrates and/or catalysis.

Two maize plastidic NADP-malic enzyme isoforms have been characterized: the bundle sheath-located photosynthetic isoform (ZmC(4)-NADP-ME) and a constitutively expressed one (Zm-nonC(4)-NADP-ME). In this work, the characterization of the first maize cytosolic NADP-ME (ZmCytNADP-ME) is presented, which transcript is exclusively found in embryo and emerging roots. ZmCytNADP-ME expression in roots decreases with development, while Zm-nonC ( 4 ) -NADP-ME increases concomitantly. On the other hand, ZmCytNADP-ME accumulation is differentially modulated by several stress conditions and shows coordination with that of Zm-nonC ( 4 ) -NADP-ME in maize young roots. Recombinant ZmCytNADP-ME displays clearly distinct kinetic parameters and metabolic regulation than the plastidic isoforms. The particular properties and the specific-expression pattern of this novel isoform suggest that it may be involved in the control of cytosolic malate levels in emerging roots, e.g. during hypoxia. ZmCytNADP-ME is phylogenetically related to other cytosolic mono and dicot NADP-MEs, and data indicate that it belongs to an ancestral unique group among plant NADP-MEs.

C(4)-specific (photosynthetic) NADP(+)-dependent malic enzyme (NADP(+)-ME) has evolved from C(3)-malic enzymes and represents a unique and specialized form, as indicated by its particular kinetic and regulatory properties. In the present paper, we have characterized maize (Zea mays L.) photosynthetic NADP(+)-ME mutants in which conserved basic residues (lysine and arginine) were changed by site-directed mutagenesis. Kinetic characterization and oxaloacetate partition ratio of the NADP(+)-ME K255I (Lys-255-->Ile) mutant suggest that the mutated lysine residue is implicated in catalysis and substrate binding. Moreover, this residue could be acting as a base, accepting a proton in the malate oxidation step. At the same time, further characterization of the NADP(+)-ME R237L mutant indicates that Arg-237 is also a candidate for such role. These results suggest that both residues may play 'back-up' roles as proton acceptors. On the other hand, Lys-435 and/or Lys-436 are implicated in the coenzyme specificity (NADP(+) versus NAD(+)) of maize NADP(+)-ME by interacting with the 2'-phosphate group of the ribose ring. This is indicated by both the catalytic efficiency with NADP(+) or NAD(+), as well as by the reciprocal inhibition constants of the competitive inhibitors 2'-AMP and 5'-AMP, obtained when comparing the double mutant K435/6L (Lys-435/436-->Ile) with wild-type NADP(+)-ME. The results obtained in the present work indicate that the role of basic residues in maize photosynthetic NADP(+)-ME differs significantly with respect to its role in non-plant MEs, for which crystal structures have been resolved. Such differences are discussed on the basis of a predicted three-dimensional model of the enzyme.

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