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C S Andreo

Publications and source records attributed to C S Andreo.

At least 19 recordsLinked to original sources
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

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.

Cellulase↗
PubMed2001

Compartmentation of photosynthesis in cells and tissues of C(4) plants.

Critical to defining photosynthesis in C(4) plants is understanding the intercellular and intracellular compartmentation of enzymes between mesophyll and bundle sheath cells in the leaf. This includes enzymes of the C(4) cycle (including three subtypes), the C(3) pathway and photorespiration. The current state of knowledge of this compartmentation is a consequence of the development and application of different techniques over the past three decades. Initial studies led to some alternative hypotheses on the mechanism of C(4) photosynthesis, and some controversy over the compartmentation of enzymes. The development of methods for separating mesophyll and bundle sheath cells provided convincing evidence on intercellular compartmentation of the key components of the C(4) pathway. Studies on the intracellular compartmentation of enzymes between organelles and the cytosol were facilitated by the isolation of mesophyll and bundle sheath protoplasts, which can be fractionated gently while maintaining organelle integrity. Now, the ability to determine localization of photosynthetic enzymes conclusively, through in situ immunolocalization by confocal light microscopy and transmission electron microscopy, is providing further insight into the mechanism of C(4) photosynthesis and its evolution. Currently, immunological, ultrastructural and cytochemical studies are revealing relationships between anatomical arrangements and photosynthetic mechanisms which are probably related to environmental factors associated with evolution of these plants. This includes interesting variations in the C(4) syndrome in leaves and cotyledons of species in the tribe Salsoleae of the family Chenopodiaceae, in relation to evolution and ecology. Thus, analysis of structure-function relationships using modern techniques is a very powerful approach to understanding evolution and regulation of the photosynthetic carbon reduction mechanisms.

Carbon Dioxide↗
PubMed2000

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.

Abscisic Acid↗
PubMed1999

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.

Enzyme Induction↗
PubMed1998

Factors affecting the oligomeric state of NADP-malic enzyme from maize and wheat tissues: a chemical crosslinking study.

The different aggregational states of maize and wheat NADP-malic enzyme as affected by pH, temperature and various metabolites have been studied by the combined use of intersubunit crosslinking and denaturing polyacrylamide gel electrophoresis. The association/dissociation equilibrium is a pH-dependent process: pH values above 8.0 promote the tetramer formation, while lowering the pH shifts the equilibria towards dimers and monomers. Below pH 6.0, most molecules exist as monomers. In the same way, the temperature governs the equilibria between the different oligomeric states. As the temperature is lowered from 42 to 0 degrees C, a progressive dissociation into dimers and monomers is observed. Excess enthalpies are negative in all cases, but the overall process demands an input of Gibb's free energy. Consequently, the protein dissociation is an entropy-driven process. The presence of Mg2+ or glycerol induces aggregation in both enzymes, while increasing the ionic strength produces the opposite effect. The results suggest that changes in the equilibria between monomer, dimer and tetramer of NADP-malic enzyme could be the molecular basis for an effective regulation of the enzyme activity in vivo.

Cross-Linking Reagents↗
PubMed1998

The interaction of shikimic acid and protein phosphorylation with PEP carboxylase from the C4 dicot Amaranthus viridis.

Shikimic acid has been described as a potent competitive inhibitor of the activity of C4 phosphoenolpyruvate carboxylase (PEPC) from Amaranthus viridis. In the present study, the effects of shikimic acid were examined further with the dephospho (dark-form) and in vitro phosphorylated forms of homogeneous PEPC from A. viridis. Kinetic analysis showed that the inhibitor effect of shikimic acid was dependent on the phosphorylation state of the enzyme. Thus, the I50 value of shikimic acid for dark-form PEPC was six times lower than that for the phosphorylated enzyme (12 vs 71 microM, respectively). When Glc6P, an activator of C4 PEPC, was present in the assay medium, the I50 value increased 2- and 3-times with the phospho and dephospho PEPC-forms, respectively. Shikimic acid also markedly decreased 32P incorporation from Mg[gamma-32P]ATP into the dark-form of C4 PEPC, but not casein, catalyzed by protein kinase A. In this way, shikimic acid mimics the behaviour of L-malate, a well-known inhibitor of PEPC, in that it decreases both the enzyme's activity and phosphorylatability. Based on these data, a possible role for shikimic acid in the regulation of PEPC activity in plants is suggested.

Adenosine Triphosphate↗
PubMed1996

Flavonoids as inhibitors of NADP-malic enzyme and PEP carboxylase from C4 plants.

The inhibitory effects of flavonoids on the activity of two photosynthetic enzymes such as phosphoenolpyruvate carboxylase (PEPCase) and NADP-dependent malic enzyme (NADP-ME) were evaluated. The glycosylation of hydroxyl groups on the flavonoids resulted in compounds that behaved as gradually weaker inhibitors with increased size of the substituent. Quercetin and baicalein showed a competitive inhibition pattern vs. NADP+ for NADP-ME, and a similar model for both flavonoids vs. phosphoenolpyruvate (PEP) was observed when tested on PEPCase. K(i) for NADP-ME inhibition at pH 7.0 were 0.83 microM and 1.54 microM for quercetin and baicalein, respectively. K(i) for PEPCase inhibition were 0.17 microM and 0.79 microM (quercetin and baicalein, respectively), indicating that these compounds are the most potent inhibitors described for this carboxylase. I50 values for these and other flavonoids were in the micromolar range. A tentative physiological role for the inhibitory effects observed on PEPCase is discussed.

Enzyme Inhibitors↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1995

Kinetic analysis of the non-phosphorylated, in vitro phosphorylated, and phosphorylation-site-mutant (Asp8) forms of intact recombinant C4 phosphoenolpyruvate carboxylase from sorghum.

Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8-->Asp) forms of purified recombinant sorghum C4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.31) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent Ki for the negative allosteric effector L-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent Ka for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1 mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and Km (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by L-malate and, most especially, Ka (Glc6P) and Ki (L-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C4-photosynthesis enzyme.

Edible Grain↗
PubMed1995

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.

Binding Sites↗
PubMed1994

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.

Disulfides↗
PubMed1993

An engineered change in the L-malate sensitivity of a site-directed mutant of sorghum phosphoenolpyruvate carboxylase: the effect of sequential mutagenesis and S-carboxymethylation at position 8.

A recombinant, site-directed mutant form of sorghum phosphoenolpyruvate carboxylase (PEPC), in which the phosphorylatable serine residue (Ser-8) was changed to cysteine (S8C), was chemically modified by iodoacetic acid and iodoacetamide for the purpose of testing the effect of introducing a negative charge at position 8. S-Carboxymethylation of the Cys-8 enzyme by iodoacetic acid decreased its sensitivity to L-malate from an I0.5 (50% inhibition) value of 0.12 to 0.35 mM at pH 7.3 when the active-site domain was protected during modification by the substrate phosphoenolpyruvate (PEP). In contrast, neither S-carboxymethylation of the wild-type enzyme nor modification of the mutant enzyme by iodoacetamide caused any change in the enzyme's sensitivity to L-malate. The modified, substrate-protected forms of the Ser-8 and S8C PEPCs had Km(total PEP) and Vmax values virtually identical to those of the unmodified control enzymes. Similar specific increases in the I0.5 value of L-malate have been reported previously for in vitro phosphorylated leaf and recombinant Ser-8 PEPCs, the site-directed mutant Asp-8 enzyme, and C4-leaf PEPC purified from light-adapted sorghum or maize (in vivo phospho-form). Therefore, these data from different but complementary experimental approaches provide convincing evidence that the effect of phosphorylation of Ser-8 on the L-malate sensitivity of sorghum C4-PEPC is caused by the introduction of negative charge into this N-terminal regulatory domain.

Amino Acid Sequence↗
PubMed1992

NADP-malic enzyme from plants.

NADP-malic enzyme functions in plant metabolism as a decarboxylase of malate in the chloroplast or cytosol. It serves as a source of CO2 for photosynthesis in the bundle sheath chloroplasts of C4 plants and in the cytosol of Crassulacean acid metabolism plants, and as a source of NADPH and pyruvate in the cytosol of various tissues. Mg2+ or Mn2+ is required as a cofactor. The enzyme has a high specificity and low Km for NADP+. It exists as a tetramer which may undergo changes in oligomerization and exhibit hysteresis. Its kinetic properties vary depending on the compartmentation and function of the enzyme. The chloroplast form in C4 plants has a high pH optimum (pH 8) under high malate, which favours the tetramer, whereas lower pH (pH 7) favours the dimer form. Generally, other forms of the enzyme, which are thought to be cytosolic, have lower pH optima than the chloroplast enzyme. In a number of cases these forms have been shown to have allosteric properties with malate as a substrate. Chemical modifications of the plant enzyme suggest sulphydryl, histidine and arginine residues are required for catalysis. Primary sequence studies on the chloroplastic enzyme from C4 plants show significant similarities to cytosolic NADP-ME in plants and animals, including a sequence motif which is indicative of the NADP+ binding site. The possible origin of the chloroplast form of the enzyme is discussed.

Amino Acid Sequence↗
PubMed1992

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.

Journal Article↗
PubMed1991

NADP(+)-malic enzyme from sugarcane leaves: structural properties studied by thermal inactivation.

The irreversible thermal inactivation of the sugarcane leaf NADP(+)-malic enzyme was studied at 50 degrees C and pH 7.0 and 8.0. Depending on the preincubation conditions, thermal inactivation followed mono- or biphasic first-order kinetics. A two-step behavior in the irreversible denaturation process was found when protein concentration was sufficiently low. The protein concentration necessary to obtain monlphasic thermal inactivation kinetics was lower at pH 8.0 than at pH 7.0. The results suggest that biphasic inactivation kinetics are the consequence of the existence of two different oligomeric forms of the enzyme (dimer and tetramer), with the dimer being more stable in regards to thermal inactivation. The effects of the substrate and essential cofactors on the thermostability and equilibrium between the dimeric and tetrameric enzyme forms were also studied. Depending on the pH, NADP+, L-malate, and Mg2+ all had a protective effect on the stability of the dimeric and tetrameric species during thermal treatment. However, these ligands showed different effects on the aggregation state of the enzyme. NADP+ and L-malate induced dissociation, especially at pH 8.0, whereas Mg2+ induced aggregation of the protein. By studying the thermal inactivation kinetics at 50 degrees C and different pH values it was observed that the equilibrium between dimers and tetramers was dramatically affected in the range of pH 7.0-8.0. These results suggest that an amino acid residue(s) in the protein with an apparent pKa value of 7.7 needs to be deprotonated to stabilize aggregation of the enzyme to the tetrameric form.

Enzyme Activation↗
PubMed1990

Isolation and sequence of an active-site peptide from maize leaf phosphoenolpyruvate carboxylase inactivated by pyridoxal 5'-phosphate.

An active-site peptide from maize (Zea mays L.) phosphoenolpyruvate carboxylase has been isolated, sequenced and identified in the primary structure following chemical modification/inactivation of the enzyme by pyridoxal 5'-phosphate and reduction with sodium borohydride. The amino acid sequence of the purified dodecapeptide is Val-Gly-Tyr-Ser-Asp-Ser-Gly-L*ys-Asp-Ala-Gly-Arg, which corresponds exactly to residues 599-610 in the deduced primary sequence of the maize-leaf enzyme. Comparative analysis of the deduced amino acid sequences of the enzyme from Escherichia coli, Anacystis nidulans and C3, C4 and Crassulacean acid metabolism plants indicates that they all contain this specific lysyl group, as well as a high degree of sequence homology flanking this species-invariant residue. This observation suggests a critical role for Lys-606 during catalysis by maize phosphoenolpyruvate carboxylase. This represents the first identification of a specific, species-invariant active-site residue in the enzyme.

Amino Acid Sequence↗