The crystal structure of the malic enzyme from Candidatus Phytoplasma reveals the minimal structural determinants for a malic enzyme.

Phytoplasmas are wall-less phytopathogenic bacteria that produce devastating effects in a wide variety of plants. Reductive evolution has shaped their genome, with the loss of many genes, limiting their metabolic capacities. Owing to the high concentration of C4 compounds in plants, and the presence of malic enzyme (ME) in all phytoplasma genomes so far sequenced, the oxidative decarboxylation of L-malate might represent an adaptation to generate energy. Aster yellows witches'-broom (Candidatus Phytoplasma) ME (AYWB-ME) is one of the smallest of all characterized MEs, yet retains full enzymatic activity. Here, the crystal structure of AYWB-ME is reported, revealing a unique fold that differs from those of `canonical' MEs. AYWB-ME is organized as a dimeric species formed by intertwining of the N-terminal domains of the protomers. As a consequence of such structural differences, key catalytic residues such as Tyr36 are positioned in the active site of each protomer but are provided by the other protomer of the dimer. A Tyr36Ala mutation abolishes the catalytic activity, indicating the key importance of this residue in the catalytic process but not in the dimeric assembly. Phylogenetic analyses suggest that larger MEs (large-subunit or chimeric MEs) might have evolved from this type of smaller scaffold by gaining small sequence cassettes or an entire functional domain. The Candidatus Phytoplasma AYWB-ME structure showcases a novel minimal structure design comprising a fully functional active site, making this enzyme an attractive starting point for rational genetic design.

Non-photosynthetic 'malic enzyme' from maize: a constituvely expressed enzyme that responds to plant defence inducers.

The characterization of a non-photosynthetic isoform of NADP-malic enzyme (NADP-ME) from maize roots, which represents nearly 7% of the total soluble protein of this tissue, was performed. The molecular properties of the purified protein, as well as the kinetic parameters determined, indicate that the NADP-ME isoform present in maize roots differs from the photosynthetic enzyme implicated in the C4 cycle, but is similar, or identical, to the enzyme previously characterized from etiolated maize leaves (Maurino, Drincovich and Andreo, Biochem. Mol. Biol. Int. 38 (1996) 239-250). A full-length ORF encoding a plastidic NADP-ME (almost identical to the maize root NADP-ME, GenBank accession number U39958) was cloned from a root cDNA library as well as isolated by reverse transcription (RT)-PCR using green leaves mRNA as template. These results indicate that root NADP-ME does not constitute a root-specific isoform, but represents a protein with a constitutive pattern of expression in plastids of the C4 plant maize. The amount of NADP-ME measured by activity, western and northern blot was modified when different stress conditions (including treatments with cellulase, fungal elicitors, jasmonate and hypoxic treatment) were applied to maize roots, indicating that the enzyme from maize roots is under transcriptional or post-transcriptional regulation by effectors related to plant defence responses. It is deduced that the induction of housekeeping genes, like non-photosynthetic NADP-ME, whose constitutive role may be the provision of reductive power in non-photosynthetic plastids, is likely to accompany the defence response.

NADP-malic enzyme from plants: a ubiquitous enzyme involved in different metabolic pathways.

NADP-malic enzyme (NADP-ME) is a widely distributed enzyme that catalyzes the oxidative decarboxylation of L-malate. Photosynthetic NADP-MEs are found in C4 bundle sheath chloroplasts and in the cytosol of CAM plants, while non-photosynthetic NADP-MEs are either plastidic or cytosolic in various plants. We propose a classification of plant NADP-MEs based on their physiological function and localization and we describe recent advances in the characterization of each isoform. Based on the alignment of amino acid sequences of plant NADP-MEs, we identify putative binding sites for the substrates and analyze the phylogenetic origin of each isoform, revealing several features of the molecular evolution of this ubiquitous enzyme.

Regulation of the expression of NADP-malic enzyme by UV-B, red and far-red light in maize seedlings.

The induction of nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) in etiolated maize (Zea mays) seedlings by UV-B and UV-A radiation, and different levels of photosynthetically active radiation (PAR, 400-700 nm) was investigated by measuring changes in activity, protein quantity and RNA levels as a function of intensity and duration of exposure to the different radiations. Under low levels of PAR, exposure to UV-B radiation but not UV-A radiation for 6 to 24 h caused a marked increase in the enzyme levels similar to that observed under high PAR in the absence of UV-B. UV-B treatment of green leaves following a 12-h dark period also caused an increase in NADP-ME expression. Exposure to UV-B radiation for only 5 min resulted in a rapid increase of the enzyme, followed by a more gradual rise with longer exposure up to 6 h. Low levels of red light for 5 min or 6 h were also effective in inducing NADP-ME activity equivalent to that obtained with UV-B radiation. A 5-min exposure to far-red light following UV-B or red light treatment reversed the induction of NADP-ME, and this effect could be eliminated by further treatment with UV-B or red light. These results indicate that physiological levels of UV-B radiation can have a positive effect on the induction of this photosynthetic enzyme. The reducing power and pyruvate generated by the activity of NADP-ME may be used for respiration, in cellular repair processes and as substrates for fatty acid synthesis required for membrane repair.

Regulation of the expression of NADP-malic enzyme by UV-B, red and far-red light in maize seedlings

The induction of nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) in etiolated maize (Zea mays) seedlings by UV-B and UV-A radiation, and different levels of photosynthetically active radiation (PAR, 400-700 nm) was investigated by measuring changes in activity, protein quantity and RNA levels as a function of intensity and duration of exposure to the different radiations. Under low levels of PAR, exposure to UV-B radiation but not UV-A radiation for 6 to 24 h caused a marked increase in the enzyme levels similar to that observed under high PAR in the absence of UV-B. UV-B treatment of green leaves following a 12-h dark period also caused an increase in NADP-ME expression. Exposure to UV-B radiation for only 5 min resulted in a rapid increase of the enzyme, followed by a more gradual rise with longer exposure up to 6 h. Low levels of red light for 5 min or 6 h were also effective in inducing NADP-ME activity equivalent to that obtained with UV-B radiation. A 5-min exposure to far-red light following UV-B or red light treatment reversed the induction of NADP-ME, and this effect could be eliminated by further treatment with UV-B or red light. These results indicate that physiological levels of UV-B radiation can have a positive effect on the induction of this photosynthetic enzyme. The reducing power and pyruvate generated by the activity of NADP-ME may be used for respiration, in cellular repair processes and as substrates for fatty acid synthesis required for membrane repair

NADP-malic enzyme isoforms in maize leaves.

Two isoforms of NADP-malic enzyme have been characterized in maize leaves. The 72 kDa-form of the protein, present mainly in etiolated maize leaves, has lower specific activity than the 62 kDa-form, which is implicated in C4 metabolism and predominates in green leaves. The larger form of the enzyme has higher Km values for NADP and malate and lower PH optimum. The antibodies raised against the 62 kDa-form of the protein react with the 72 kDa-form. Steady state levels of NADP-malic enzyme, as measured by the amount of protein and activity, increase several-fold when dark-grown maize seedlings are illuminated. This increase in protein is about 13-fold for the 62 kDa-form of the enzyme, while the 72 kDa-form remains practically constant after a transient increase. Northern blot analysis using a specific probe against the 62 kDa-form of the enzyme, reveals the increase of a 2.2 kb mRNA during greening. Southern hybridization analysis with genomic DNA suggests the presence of more than one gene encoding NADP-malic enzyme in maize. In this paper we provide biochemical and inmunological evidence suggesting that both isoforms are closely related and that the 72 kDa-form is also present in low levels in mature green leaves.

NADP-malic enzyme from maize leaves: a fluorescence study.

NADP-malic enzyme from maize leaves is covalently labeled with a fluorescent-SH reactive probe eosin-5-maleimide (EMA), which reacts with groups that are totally protected by NADP against inactivation. The comparison of the emission fluorescence spectra of the native and the modified enzyme suggests the proximity of the fluorescent groups of the native enzyme (probably tryptophanyl groups) and the EMA modified residues. Intrinsic fluorescence quenching studies shows that NADP is the only substrate capable to interact with the fluorescent excited groups of the enzyme, while Mg2+ is able to increase this interaction. Quenching studies of EMA-bound fluorescence shows that the NADP-binding site was modified and thus uncapable of further interaction with the nucleotide. When the results of protection studies are combined with those of extrinsic quenching experiments, we must conclude that EMA reacts with sulfhydryl groups that are involved in the NADP-binding site of the enzyme.

Redox regulation of maize NADP-malic enzyme by thiol-disulfide interchange: effect of reduced thioredoxin on activity.

Incubation of C4 NADP-malic enzyme from maize leaves with the oxidant o-iodosobenzoate leads to the reversible and complete inactivation of the enzyme. The time-course of inactivation is biphasic with the rate depending on the o-iodosobenzoate concentration. The inactivation is partially prevented by L-malate, NADP and Mg2+ alone, while NADP plus Mg2+ afford total protection. The complete reversal of the inactivation by the reductive agents dithiothreitol and 2-mercaptoethanol suggests that the modification of the enzyme by o-iodosobenzoate occurs concomitant with the oxidation of one or more pairs of sulfhydryl groups to the disulfide state, producing a conformationally altered form of the protein or directly modifying the active site. Titration of free thiol groups before and after inactivation of maize malic enzyme by o-iodosobenzoate shows a decrease in the accessible groups from 7 to 5, suggesting inactivation is accompanied by oxidation of two vicinal thiols. The oxidized form of the enzyme is rapidly reactivated by incubation with chemical and photochemically reduced thioredoxin in vitro, while the 'dark' activity of the enzyme is enhanced to the level of the 'light' activity by dithiothreitol. This evidence suggests that a reversible reduction and oxidation of disulfide bonds may take place during the regulation of the enzyme, indicating that the redox state of the disulfide bonds of C4 NADP-malic enzyme from maize leaves is important for the expression of maximal catalytic activity.

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves.

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. The inactivation by mono and bifunctional maleimides was partially prevented by Mg(2+) and l-malate, and NADP prevented the inactivation almost totally. Determination of the number of reactive sulfhydryl groups of the native enzyme with [(3)H]NEM in the absence or presence of NADP showed that inactivation occurred concomitantly with the modification of two cysteinyl residues per enzyme monomer. The presence of these two essential residues was confirmed by titration of sulfhydryl groups with [(3)H]NEM in the enzyme previously modified by o-phenylenebismaleimide in the absence or presence of NADP.