Fumarate and cytosolic pH as modulators of the synthesis or consumption of C4 organic acids through NADP-malic enzyme in Arabidopsis thaliana

Arabidopsis thaliana is a plant species that accumulates high levels of organic acids and uses them as carbon, energy and reducing power sources. Among the enzymes that metabolize these compounds, one of the most important ones is malic enzyme (ME). A. thaliana contains four malic enzymes (NADP-ME 1–4) to catalyze the reversible oxidative decarboxylation of malate in the presence of NADP. NADP-ME2 is the only one located in the cell cytosol of all Arabidopsis organs providing most of the total NADP-ME activity. In the present work, the regulation of this key enzyme by fumarate was investigated by kinetic assays, structural analysis and a site-directed mutagenesis approach. The final effect of this metabolite on NADP-ME2 forward activity not only depends on fumarate and substrate concentrations but also on the pH of the reaction medium. Fumarate produced an increase in NADP-ME2 activity by binding to an allosteric site. However at higher concentrations, fumarate caused a competitive inhibition, excluding the substrate malate from binding to the active site. The characterization of ME2-R115A mutant, which is not activated by fumarate, confirms this hypothesis. In addition, the reverse reaction (reductive carboxylation of pyruvate) is also modulated by fumarate, but in a different way. The results indicate pH-dependence of the fumarate modulation with opposite behavior on the two activities analyzed. Thereby, the coordinated action of fumarate over the direct and reverse reactions would allow a precise and specific modulation of the metabolic flux through this enzyme, leading to the synthesis or degradation of C₄ compounds under certain conditions. Thus, the physiological context might be exerting an accurate control of ME activity in planta, through changes in metabolite and substrate concentrations and cytosolic pH.     

Maize recombinant non-C<Subscript>4</Subscript> NADP-malic enzyme: A novel dimeric malic enzyme with high specific activity

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 C₄-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-C₄ 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 invivo. 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 ZmChlMe2gene and the likely precursor to the evolution of the photosynthetic C₄ NADP-ME) and the 62 kDa isoform (implicated in C₄ 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 C₄ isoenzyme in maize.     

A novel biotin protein required for reductive carboxylation of 2-oxoglutarate by isocitrate dehydrogenase in Hydrogenobacter thermophilus TK-6

Isocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters. Notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2-oxoglutarate. The carboxylation reaction required the addition of cell extract and ATP-Mg, suggesting the existence of additional carboxylation factor(s). Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides. N-terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation. Pyruvate carboxylase was also purified, but was not active in stimulating isocitrate dehydrogenase. Isocitrate dehydrogenase, the novel biotin protein, ATP-Mg and NADH were essential for the reductive carboxylation of 2-oxoglutarate. These observations indicate that the novel biotin protein is an ATP-dependent factor, which is involved in the reverse (carboxylating) reaction of isocitrate dehydrogenase.     

Identification of domains involved in tetramerization and malate inhibition of maize C4-NADP-malic enzyme

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.     

Maize C4 and non-C4 NADP-dependent malic enzymes are encoded by distinct genes derived from a plastid-localized ancestor

NADP-dependent malic enzymes (NADP-ME; EC1.1.1.40) have been implicated in a wide range of metabolic pathways in the plastids and cytosol of plant cells. In maize, an NADP-ME type C4 plant, the most abundant NADP-ME form is the chloroplastic leaf isoform that delivers CO₂ intracellularly to ribulose bisphosphate carboxylase (RuBPCase). A second NADP-ME isoform predominates in maize roots and exhibits distinct C3-like enzymatic characteristics. We show that the C3-like isoform is encoded by a pair of nearly identical genes that encode precursor proteins with functional chloroplast transit peptides. Using RT-PCR, we also show that the messages encoding the C4 and C3-like NADP-ME isoforms are differentially regulated with respect to the developmental stage of the leaf, light conditions, and tissue type. Based on these characteristics and on sequence comparison of ME families in other species, we propose a scheme for the origin of the C4-specific NADP-ME gene.     

A comprehensive analysis of the NADP-malic enzyme gene family of Arabidopsis

The Arabidopsis (Arabidopsis thaliana) genome contains four genes encoding putative NADP-malic enzymes (MEs; AtNADP-ME1-ME4). NADP-ME4 is localized to plastids, whereas the other three isoforms do not possess any predicted organellar targeting sequence and are therefore expected to be cytosolic. The plant NADP-MEs can be classified into four groups: groups I and II comprising cytosolic and plastidic isoforms from dicots, respectively; group III containing isoforms from monocots; and group IV composed of both monocots and dicots, including AtNADP-ME1. AtNADP-MEs contained all conserved motifs common to plant NADP-MEs and the recombinant isozymes showed different kinetic and structural properties. NADP-ME2 exhibits the highest specific activity, while NADP-ME3 and NADP-ME4 present the highest catalytic efficiency for NADP and malate, respectively. NADP-ME4 exists in equilibrium of active dimers and tetramers, while the cytosolic counterparts are present as hexamers or octamers. Characterization of T-DNA insertion mutant and promoter activity studies indicates that NADP-ME2 is responsible for the major part of NADP-ME activity in mature tissues of Arabidopsis. Whereas NADP-ME2 and -ME4 are constitutively expressed, the expression of NADP-ME1 and NADP-ME3 is restricted by both developmental and cell-specific signals. These isoforms may play specific roles at particular developmental stages of the plant rather than being involved in primary metabolism.     

Induction of a C(4)-like mechanism of CO(2) fixation in Egeria densa, a submersed aquatic species

The expression of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme (NADP-ME) in Egeria densa leaves was studied under low temperature and light (LTL) following incubation under high temperature and light (HTL), conditions previously shown to induce high and low CO(2) compensation points, respectively. Transfer from LTL to HTL conditions induced increases in the activities and amounts of both enzymes. One NADP-ME isoform was observed in induced and uninduced samples. Two isoforms of PEPC were expressed, with the lower M(r) isoform being induced by HTL. NADP-ME showed properties similar to those of the isoform in C(3) species. The inducible PEPC isoform has a low K(m) for both substrates. PEPC kinetic and regulatory properties (V(max) and K(m) for phosphoenolpyruvate, and I(50) for L-malate) are different in samples taken in the dark from those in the light, indicating that some modification of PEPC may be occurring during the day. Finally, abscisic acid induced the expression of PEPC and NADP-ME in a manner similar to temperature induction, except that the activities of both PEPC isoforms were increased. A different signaling system may exist in this species in response to high temperature or abscisic acid, both of which induce changes in photosynthetic metabolism.     

The mechanisms of reductive carboxylation reactions. Carbon dioxide or bicarbonate as substrate of nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase and `malic'' enzyme

1. A simple kinetic method was devised to show whether dissolved CO(2) or HCO(3)- ion is the substrate in enzyme-catalysed carboxylation reactions. 2. The time-course of the reductive carboxylation of 2-oxoglutarate by NADPH, catalysed by isocitrate dehydrogenase, was studied by a sensitive fluorimetric method at pH7.3 and pH6.4, with large concentrations of substrate and coenzyme and small carbon dioxide concentrations. 3. Reaction was initiated by the addition of carbon dioxide in one of three forms: (i) as the dissolved gas in equilibrium with bicarbonate; (ii) as unbuffered bicarbonate solution; (iii) as the gas or as an unbuffered solution of the gas in water. Different progress curves were obtained in the three cases. 4. The results show that dissolved CO(2) is the primary substrate of the enzyme, and that HCO(3)- ion is at best a very poor substrate. The progress curves are in quantitative agreement with this conclusion and with the known rates of the reversible hydration of CO(2) under the conditions of the experiments. The effects of carbonic anhydrase confirm the conclusions. 5. Similar experiments on the reductive carboxylation of pyruvate catalysed by the ;malic' enzyme show that dissolved CO(2) is the primary substrate of this enzyme also. 6. The results are discussed in relation to the mechanisms of these enzymes, and the effects of pH on the reactions. 7. The advantages of the method and its possible applications to other enzymes involved in carbon dioxide metabolism are discussed.     

Characterization of the NADP malic enzyme gene family in the facultative,single-cell C<Subscript>4</Subscript> monocot <Emphasis Type="Italic">Hydrilla verticillata</Emphasis>

Hydrilla verticillata has a facultative single-cell system that changes from C₃ to C₄ photosynthesis. A NADP⁺-dependent malic enzyme (NADP-ME) provides a high [CO₂] for Rubisco fixation in the C₄ leaf chloroplasts. Of three NADP-ME genes identified, only hvme1 was up-regulated in the C₄ leaf, during the light period, and it possessed a putative transit peptide. Unlike obligate C₄ species, H. verticillata exhibited only one plastidic isoform that may perform housekeeping functions, but is up-regulated as the photosynthetic decarboxylase. Of the two cytosolic forms, hvme2 and hvme3, the latter exhibited the greatest expression, but was not light-regulated. The mature isoform of hvme1 had a pI of 6.0 and a molecular mass of 64 kD, as did the recombinant rHVME1m, and it formed a tetramer in the chloroplast. The recombinant photosynthetic isoform showed intermediate characteristics between isoforms in terrestrial C₃ and C₄ species. The catalytic efficiency of rHVME1m was four-fold higher than the cytosolic rHVME3 and two-fold higher than recombinant cytosolic isoforms of rice, but lower than plastidic forms of maize. The K _m (malate) of 0.6 mM for rHVME1 was higher than maize plastid isoforms, but four-fold lower than found with rice. A comprehensive phylogenetic analysis of 25 taxa suggested that chloroplastic NADP-ME isoforms arose from four duplication events, and hvme1 was derived from cytosolic hvme3. The chloroplastic eudicot sequences were a monophyletic group derived from a cytosolic clade after the eudicot and monocot lineages separated, while the monocots formed a polyphyletic group. The findings support the hypothesis that a NADP-ME isoform with specific and unusual regulatory properties facilitates the functioning of the single-cell C₄ system in H. verticillata. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Maize C4 NADP-malic enzyme. Expression in Escherichia coli and characterization of site-directed mutants at the putative nucleoside-binding sites

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.     

Human 17beta-hydroxysteroid dehydrogenases types 1, 2, and 3 catalyze bi-directional equilibrium reactions, rather than unidirectional metabolism, in HEK-293 cells

Human 17beta-hydroxysteroid dehydrogenases (17betaHSDs) catalyze the interconversion of weak and potent androgen and estrogen pairs. Although the reactions using purified enzymes can be driven in either direction, these enzymes appear to function unidirectionally in intact cells: only reductive reactions for 17betaHSD1 and 17beta HSD3 and only oxidative reactions for 17betaHSD2. We show that, after exhaustive incubations with either 17beta-hydroxy- or 17-ketosteroid, the medium for HEK-293 cells expressing 17betaHSD1 or 17betaHSD3 contains a 92:8 ratio of reduced:oxidized steroid. Similarly, 17betaHSD2 yields a >95:5 ratio of oxidized:reduced steroids for both androgens and estrogens. Dual-isotope kinetic measurements show that the rates of the forward and reverse reactions are identical at these functional equilibrium states in intact cells for all three 17betaHSD isoforms, and these rates are much faster than those estimated from single-isotope flux studies. Mutation L36D converts 17betaHSD1 to an oxidative enzyme in intact cells, reversing the equilibrium distribution of estradiol:estrone to 5:95; however, the rates of the forward and reverse reactions at equilibrium are equal and comparable to those of the wild-type enzymes. The co-expression of 17betaHSD2 paradoxically increases the potency of estrone in transactivation assays, demonstrating the physiological relevance of "backwards" metabolism to estradiol. We conclude that 17betaHSD types 1, 2, and 3 catalyze both oxidative and reductive reactions in HEK-293 cells at intrinsic rates that are much faster than those estimated from single-isotope studies. These 17betaHSD isoforms do not drive steroid flux in one direction but rather may achieve functional equilibria in intact cells, reflecting thermodynamically driven steroid distributions.     

Carboxylation and anaplerosis in neurons and glia

Anaplerosis, or de novo formation of intermediates of the tricarboxylic acid (TCA) cycle, compensates for losses of TCA cycle intermediates, especially α-ketoglutarate, from brain cells. Loss of α-ketoglutarate occurs through release of glutamate and GABA from neurons and through export of glutamine from glia, because these amino acids are α-ketoglutarate derivatives. Anaplerosis in the brain may involve four different carboxylating enzymes: malic enzyme, phosphoenopyruvate carboxykinase (PEPCK), propionyl-CoA carboxylase, and pyruvate carboxylase. Anaplerotic carboxylation was for many years thought to occur only in glia through pyruvate carboxylase; therefore, loss of transmitter glutamate and GABA from neurons was thought to be compensated by uptake of glutamine from glia. Recently, however, anaplerotic pyruvate carboxylation was demonstrated in glutamatergic neurons, meaning that these neurons to some extent can maintain transmitter synthesis independently of glutamine. Malic enzyme, which may carboxylate pyruvate, was recently detected in neurons. The available data suggest that neuronal and glial pyruvate carboxylation could operate at as much as 30% and 40–60% of the TCA cycle rate, respectively. Cerebral carboxylation reactions are probably balanced by decarboxylation reactions, because cerebral CO₂ formation equals O₂ consumption. The finding of pyruvate carboxylation in neurons entails a major revision of the concept of the glutamine cycle.     

Characterization of five catalytic activities associated with the NADPH:2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate] oxidoreductase/carboxylase of the Xanthobacter strain Py2 epoxide carboxylase system

The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by carboxylation to acetoacetate. Epoxypropane carboxylation is a minimetabolic pathway that requires four enzymes, NADPH, NAD(+), and coenzyme M (CoM; 2-mercaptoethanesulfonate) and occurs with the overall reaction stoichiometry: epoxypropane + CO(2) + NADPH + NAD(+) + CoM --> acetoacetate + H(+) + NADP(+) + NADH + CoM. The terminal enzyme of the pathway is NADPH:2-ketopropyl-CoM [2-(2-ketopropylthio)ethanesulfonate] oxidoreductase/carboxylase (2-KPCC), an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase family of enzymes and that catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-CoM to form acetoacetate and CoM according to the reaction: 2-ketopropyl-CoM + NADPH + CO(2) --> acetoacetate + NADP(+) + CoM. In the present work, 2-KPCC has been characterized with respect to the above reaction and four newly discovered partial reactions of relevance to the catalytic mechanism, and each of which requires the formation of a stabilized enolacetone intermediate. These four reactions are (1) NADPH-dependent cleavage and protonation of 2-ketopropyl-CoM to form NADP(+), CoM, and acetone, a reaction analogous to the physiological reaction but in which H(+) is the electrophile; (2) NADP(+)-dependent synthesis of 2-ketopropyl-CoM from CoM and acetoacetate, the reverse of the physiologically important forward reaction; (3) acetoacetate decarboxylation to form acetone and CO(2); and (4) acetoacetate/(14)CO(2) exchange to form (14)C(1)-acetoacetate and CO(2). Acetoacetate decarboxylation and (14)CO(2) exchange occurred independent of NADP(H) and CoM, demonstrating that these substrates are not central to the mechanism of enolate generation and stabilization. 2-KPCC did not uncouple NADPH oxidation or NADP(+) reduction from the reactions involving cleavage or formation of 2-ketopropyl-CoM. N-Ethylmaleimide inactivated the reactions forming/using 2-ketopropyl-CoM but did not inactivate acetoacetate decarboxylation or (14)CO(2) exchange reactions. The biochemical characterization of 2-KPCC and the associated five catalytic activities has allowed the formulation of an unprecedented mechanism of substrate activation and carboxylation that involves NADPH oxidation, a redox active disulfide, thiol-mediated reductive cleavage of a C-S thioether bond, the formation of a CoM:cysteine mixed disulfide, and enolacetone stabilization.     

The pH dependence of the reductive carboxylation of pyruvate by malic enzyme

The maximum velocity of the malic enzyme (L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) reductive carboxylation of pyruvate and V/KCO2 are pH-independent from pH 5.5 to pH 8.5. V/K for pyruvate exhibits pK values values of 6.50 +/- 0.25 and 7.25 +/- 0.25. These data suggest that the binding of pyruvate locks the protonation state of enzyme. In addition, the pK values are within experimental error identical for the pH dependence of V/Kmalate and V/Kpyruvate. Thus, the catalytic groups appear to have reverse protonation states in the two reaction directions. The ratio of (V/Kmalate)/(V/Kpyruvate) is 100, suggesting that the protonation state of enzyme is optimum in the malate oxidative decarboxylation direction. Thus, the group with a pK of about 6 is unprotonated and the group with a pK of 7.5 is protonated for malate decarboxylation, and the opposite is true for pyruvate reductive carboxylation.     

The M2-type isoenzyme of pyruvate kinase phosphorylates prothymosin α in proliferating lymphocytes

Prothymosin α (ProTα) is a multifunctional protein that, in mammalian cells, is involved in nuclear metabolism through its interaction with histones and that also has a cytosolic role as an apoptotic inhibitor. ProTα is phosphorylated by a protein kinase (ProTαK), the activity of which is dependent on phosphorylation. ProTα phosphorylation also correlates with cell proliferation. Mass spectrometric analysis of ProTαK purified from human tumor lymphocytes (NC37 cells) enabled us to identify this enzyme as the M2-type isoenzyme of pyruvate kinase. A study on the relationship between ProTαK activity and pyruvate kinase isoforms in NC37 cells and in other cell types confirmed that the M2 isoform is the enzyme responsible for ProTαK activity in proliferating cells. Yet, about 10% of the cellular pool of the M2 isoform shows specific affinity for ProTα and is responsible for ProTαK activity. This pool of M2 protein possesses no observable pyruvate kinase activity and changes its responses to various effectors of pyruvate kinase activity; however, these responses to PK effectors are maintained by the main cellular fraction containing the M2 isoform. Acquisition of ProTαK activity by M2 seems to be due to the phosphorylation of serine and threonine residues, which, besides being essential for its catalytic activity, induces a trimeric association of ProTαK. This association can be shifted to a tetrameric form by fructose 1, 6-bisphosphate, which results in a decrease in ProTαK activity.     

Unidirectional inhibition and activation of "malic'' enzyme of Solanum tuberosum by meso-tartrate.

A kinetic study of "malic' enzyme (EC 1.1.1.40) from potato suggests that the mechanism is Ordered Bi Ter with NADP+ binding before malate, and NADPH binding before pyruvate and HCO3-. The analysis is complicated by the non-linearity that occurs in some of the plots. meso-Tartrate is shown to inhibit the oxidative decarboxylation of malate but to activate the reductive carboxylation of pyruvate. To explain these unidirectional effects it is suggested that the control site of "malic' enzyme binds organic acids (including meso-tartrate) which activate the enzyme. meso-Tartrate, however, competes with malate for the active site and thus inhibits the oxidative decarboxylation of malate. Because meso-tartrate does not compete effectively with pyruvate for enzyme-NADPH, its binding at the control site leads to a stimulation of the carboxylation of pyruvate. A similar explanation is advanced for the observation that malic acid stimulates its own synthesis.     

Mitochondrial NADP-dependent malic enzyme of cod heart. Rate of forward and reverse reaction

The mitochondrial NADP-dependent malic enzyme (EC 1.1.1.40) was purified about 300-fold from cod Gadus morhua heart to a specific activity of 48 units (mumol/min)/mg at 30 degrees C. The possibility of the reductive carboxylation of pyruvate to malate was studied by determination of the respective enzyme properties. The reverse reaction was found to proceed at about five times the velocity of the forward rate at a pH 6.5. The Km values determined at pH 7.0 for pyruvate, NADPH and bicarbonate in the carboxylation reaction were 4.1 mM, 15 microM and 13.5 mM, respectively. The Km values for malate, NADP and Mn2+ in the decarboxylation reaction were 0.1 mM, 25 microM and 5 microM, respectively. The enzyme showed substrate inhibition at high malate concentrations for the oxidative decarboxylation reaction at pH 7.0. Malate inhibition suggests a possible modulation of cod heart mitochondrial NADP-malic enzyme by its own substrate. High NADP-dependent malic enzyme activity found in mitochondria from cod heart supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate.