Regulation of C4 photosynthesis: regulation of activation and inactivation of NADP-malate dehydrogenase by NADP and NADPH

Inactive NADP-malate dehydrogenase (disulfide form) from chloroplasts of Zea mays is activated by reduced thioredoxin while the active enzyme (dithiol form) is inactivated by incubation with oxidized thioredoxin. This reductive activation of NADP-malate dehydrogenase is inhibited by over 95% in the presence of NADP and the Kd for this interaction of NADP with the inactive enzyme is about 3 microM. Other substrates of the enzyme (malate, oxaloacetate, or NADPH) do not effect the rate of enzyme activation but NADPH can reverse the inhibitory effect of NADP. It appears that NADPH (Kd = 250 microM) and NADP (Kd = 3 microM) compete for the same site, presumably the coenzyme-binding site at the active centre. Apparently the enzyme . NADP binary complex cannot be reduced by thioredoxin whereas the enzyme . NADPH complex is reduced at the same rate as is the free enzyme. Similarly the oxidative inactivation of reduced NADP-malate dehydrogenase is inhibited by up to 85% by NADP and NADPH completely reverses this inhibition. The Kd values of the active-reduced enzyme for NADP and NADPH were both estimated to be 30 microM. From these data a model was constructed which predicts how changing NADPH/NADP levels in the chloroplast might change the steady-state level of NADP-malate dehydrogenase activity. The model indicates that at any fixed ratio of reduced to oxidized thioredoxin high proportions of active NADP-malate dehydrogenase and, hence, high rates of oxaloacetate reduction, can only occur with very high NADPH/NADP ratios.     

Regulation of NADP-malate dehydrogenase in C4 plants: effect of varying NADPH to NADP ratios and thioredoxin redox state on enzyme activity in reconstituted systems

Activation and inactivation of NADP-malate dehydrogenase purified from Zea mays leaves were followed in a reconstituted system provided with thioredoxin poised in various redox states with dithiothreitol. The initial rate of activation or inactivation of NADP-malate dehydrogenase was proportional to the concentration of reduced or oxidized thioredoxin, respectively. The rate of inactivation was about 16 times that for activation at pH 7.4. Both activities increased when the pH was increased from 7.4 to 8.0. The redox potentials (E'0, pH 7) for the dithiol-disulfide systems of thioredoxin and NADP-malate dehydrogenase were estimated to be about -0.30 and -0.33 V, respectively. As would be predicted from these values, high proportions of active malate dehydrogenase were developed only in the presence of very high ratios of reduced to oxidized thioredoxin. Similarly, when pyridine nucleotide was included, a high degree of activation of malate dehydrogenase was only observed with high NADPH/NADP ratios. These results confirm predictions based on models developed in earlier studies that the NADPH to NADP ratio as well as the thioredoxin redox state may be critical in determining the level of NADPH-malate dehydrogenase activity in vivo.     

Gas exchange of two CAM species of the genus Cissus (vitaceae) differing in morphological features

NADP-malate dehydrogenase extracted from darkened leaves of the C₃ plants pea, barley, wheat and spinach was activated by reduced glutathione, a monothiol, as well as by dithiothreitol (DTT). However, in the C₄ plants maize and Flaveria trinervia, only dithiothreitol could effectively activate the enzyme. There was no activation of the maize enzyme and little or no activation of the F. trinervia enzyme by glutathione. The failure of glutathione to activate NADP-MDH in leaf extracts of maize and F. trinervia may indicate there is some difference in disulfide groups of the protein compared to the C₃ plant enzyme. Both DTT and glutathione could activate NADP-malate dehydrogenase in a partially purified enzyme preparation from pea leaves with or without addition of partially purified thioredoxin. However, the required concentration of reductant was lower with addition of thioredoxin than in its absence. In extracts of C₃ species and the partially purified pea enzyme the level of activation after 40 to 60 min under aerobic conditions was higher (up to twofold) with DTT than with glutathione. Under anaerobic conditions, the initial rate of activation was about twice as high with DTT as with glutathione, but the total activation after 40 to 60 min was similar. Ascorbate was totally ineffective as a reducing agent in activating NADP-MDH from C₃ or C₄ plants, possibly due to its more positive redox potential.Abbreviations Chl Chlorophyll - DTT Dithiothreitol - GSH Reduced Glutathione - NADP-MDH NADP-malate Dehydrogenase     

Site-directed mutagenesis reveals the involvement of an additional thioredoxin-dependent regulatory site in the activation of recombinant sorghum leaf NADP-malate dehydrogenase.

The chloroplastic enzyme NADP-malate dehydrogenase is activated by a reversible thiol/disulfide interchange with reduced thioredoxin. Its target disulfide bridge is considered to be located at the amino terminus. To further substantiate the regulatory role of this disulfide, site-directed mutagenesis has been used to replace each or both of the amino-terminal cysteines of the sorghum leaf NADP-malate dehydrogenase, expressed in Escherichia coli, by serines. A truncation mutant lacking the amino terminus has also been produced. Surprisingly, the mutant proteins still required activation by reduced thioredoxin. However, their activation was almost instantaneous, whereas the native enzyme reached full activity after a 10-20 min preincubation. The 8 1/2 for reduced thioredoxin was decreased 2-fold in the mutants, but their Km values for NADPH and oxaloacetate did not change significantly. The inhibition of activation by NADP and inhibition of activity by thiol-derivatizing agents were also retained. These results are interpreted as an indication that two thioredoxin-dependent reduction steps are involved in NADP-dependent malate dehydrogenase light activation, hence that two disulfides per monomer participate in the process. The overall activation rate would depend on a conformational change following the reduction of the amino-terminal disulfide bridge. The amino terminus also plays a role in the dimerization of the protein.     

Enzyme regulation in C4 photosynthesis: Mechanism of activation of NADP-malate dehydrogenase by reduced thioredoxin

The mechanism of activation of thioredoxin-linked NADP-malate dehydrogenase was investigated by using ¹⁴C-iodoacetate and ¹⁴C-dansylated thioredoxin m, and Sepharose affinity columns (thioredoxin m, NADP-malate dehydrogenase) as probes to monitor enzyme sulfhydryl status and enzyme-thioredoxin interaction. The data indicate that NADP-malate dehydrogenase, purified to homogeneity from corn leaves, is activated by a net transfer of reducing equivalents from thioredoxin m, reduced by dithiothreitol, to enzyme disulfide groups, thereby yielding oxidized thioredoxin m and reduced enzyme. The appearance of new sulfhydryl groups that accompanies the activation of NADP-malate dehydrogenase appears to involve a structural change that is independent of the formation of a stable complex between the enzyme and reduced thioredoxin m. The data are consistent with the conclusion that oxygen promotes deactivation of NADP-malate dehydrogenase through oxidation of SH groups on reduced thioredoxin and on the reduced (activated) enzyme.     

Regulation of NADP-malate dehydrogenase in C4 plants: relationship among enzyme activity, NADPH to NADP ratios, and thioredoxin redox states in intact maize mesophyll chloroplasts

NADP-malate dehydrogenase activity, the ratio of NADPH to NADP, and thioredoxin redox state in Zea mays chloroplasts were determined after various treatments. Following transfer from dark to light, NADP-malate dehydrogenase was activated more than 20-fold within 10 min while the proportion of pyridine nucleotide as NADPH increased from about 25 to 90%, and the proportion of thioredoxin in the reduced form increased from 20 to more than 90%, in less than 1 min. After transfer back to the dark, NADPH levels dropped very rapidly to the initial values recorded before illumination, while enzyme activity and reduced thioredoxin levels decreased more slowly. Addition of oxaloacetate or 3-phosphoglycerate to illuminated chloroplasts results in a decrease of about 70% in the activity of NADP-malate dehydrogenase, a 30% decrease in the level of NADPH, and a 25% decrease in the reduced thioredoxin content. Adding dihydroxyacetone phosphate and pyruvate had no effect. These results are considered in relation to the hypothesis that NADP-malate dehydrogenase activity in chloroplasts may be determined by factors regulating the ratio of NADPH to NADP as well as those influencing the redox state of thioredoxin.     

Activation of Chloroplast NADP-linked Glyceraldehyde-3-Phosphate Dehydrogenase by the Ferredoxin/Thioredoxin System

NADP-glyceraldehyde-3-P dehydrogenase of spinach (Spinacia oleracea) chloroplasts was activated by thioredoxin that was reduced either photochemically with ferredoxin and ferredoxin-thioredoxin reductase or chemically with dithiothreitol. The activation process that was observed with the soluble protein fraction from chloroplasts and with the purified regulatory form of the enzyme was slow relative to the rate of catalysis. The NAD-linked glyceraldehyde-3-P dehydrogenase activity that is also present in chloroplasts and in the purified enzyme preparation was not affected by reduced thioredoxin.

When activated by dithiothreitol-reduced thioredoxin, the regulatory form of NADP-glyceraldehyde-3-P dehydrogenase was partly deactivated by oxidized glutathione. The enzyme activated by photochemically reduced thioredoxin was not appreciably affected by oxidized glutathione. The results suggest that although it resembles other regulatory enzymes in its requirements for light-dependent activation by the ferredoxin/thioredoxin system, NADP-glyceraldehyde-3-P dehydrogenase differs in its mode of deactivation and in its capacity for activation by enzyme effectors independently of thioredoxin.

     

The dark (oxidized) form of the light-activatable NADP-malate dehydrogenase from pea chloroplasts is catalytically active in the presence of guanidine-HCl

The dark form of NADP-malate dehydrogenase from pea leaves which has been shown to contain one disulfide bridge per subunit does not exhibit any catalytic activity in the absence of thiol reducing agents. Upon reduction of these disulfide bridges the enzyme becomes catalytically active. In this presentation, however, it is shown that the oxidized dark form of NADP-malate dehydrogenase becames catalytically competent when assayed in the presence of 200–250 mM guanidine-HCl. This guanidine-dependent activity of the oxidized enzyme is characterized by higher apparent K_m values for the substratres as compared to the reduced enzyme, but is still specific for NADPH. Up to 25% of the V_maxOf the reduced enzyme was obtained for the oxidized guanidine-activated NADP-malate dehydrogenase. The results suggest that the reduction of the regulatory disulfide is not essential for catalytic activity.     

Chloroplast glucose-6-phosphate dehydrogenase: Km shift upon light modulation and reduction

Illumination of intact chloroplasts and treatment of chloroplast stroma with dithiothreitol (DTT) both inactivate glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) to less than 10% apparent activity when assayed under standard conditions. Illumination of intact protoplasts and incubation of leaf extract with DTT inactivate about 25-35% of the total G6PDH activity. In the leaf extract, however, further loss of activity is observed if NADP is absent. Light- and DTT-inactivated chloroplast G6PDH can be reactivated by oxidation with sodium tetrathionate or the thiol oxidant diamide. Chloroplast G6PDH is as sensitive toward reductive enzyme modulation in a stromal extract as are other light/dark modulated enzymes, e.g., NADP-malate dehydrogenase. Also, glutathione, provided it is kept reduced, is sufficient to cause inactivation. Light- and DTT-induced inactivation are shown to be due to a Km shift with respect to glucose-6-phosphate (G6P) from 1 to 35 and 43 mM, respectively, and with respect to NADP from 10 to 50 microM without any significant change of the Vmax. NADPH competitively (NADP) inhibits the enzyme (Ki = 8 microM). Reactivation by oxidation can be explained by an enhanced affinity of the oxidized enzyme toward G6P and NADP. The pH optimum of the reduced enzyme is more in the alkaline region (pH 9-9.5) as compared to that of the oxidized form (pH 8.0). The presence of 30 mM phosphate causes a shift of 0.5 to 1.0 pH unit into the alkaline region for both forms.     

On the Mechanism of Activation by Light of the NADP-dependent Malate Dehydrogenase in Spinach Chloroplasts

With intact spinach (Spinacia oleracea L. cv. Vital R) chloroplasts, the activity of the NADP-dependent malate dehydrogenase after activation by light was 30 micromoles of malate formed per milligram of chlorophyll per hour; an identical rate of O₂ evolution was obtained upon oxaloacetate reduction by the intact plastids. However, when the activity of NADP-dependent malate dehydrogenase was measured subsequently to maximal activation of the enzyme by dithiothreitol (DTT) an average rate of 113 micromoles per milligram of chlorophyll per hour was obtained. When membranes and stroma were separated after osmotic disruption of the chloroplasts, 28% of NADP-dependent malate dehydrogenase activity inducible by DTT was found with the membranes and 72% was found in the stromal fraction. The membrane-associated portion of the enzyme corresponds well with the activity achieved after activation by light. About 64% of an activator system was found to be associated also with the membrane fraction. Washing the membranes with buffer removed more activator than enzyme. However, both were removed almost completely by ethylenediaminetetraacetate. It was concluded that both a portion of the enzyme and the total activator system are associated with the chloroplast membranes in vivo and that the activator is more loosely bound than the enzyme. A model describing the partial activation of chloroplastic NADP-dependent malate dehydrogenase by light and the total activation by DTT is presented.     

NADP regulates the light activation of NADP-dependent malate dehydrogenase

The chloroplastic NADP-dependent malate-dehydrogenase (EC 1.1.1.82) activity is modulated by light and dark. The enzyme is activated upon illumination of intact or broken chloroplasts or by incubation with dithiothreitol, whereas dark has the opposite effect. The present communication shows an additional regulation of the light modulation: in isolated intact pea chloroplasts, light activation was inhibited in the presence of electron acceptors such as sodium bicarbonate, 3-phosphoglycerate or oxaloacetate, which consume NADPH₂ and produce NADP. With broken chloroplasts, addition of NADP resulted in a pronounced lag phase of NADP-dependent malate dehydrogenase light activation, while NADPH₂ was without any effect. The extent of the lag phase was correlated to the amount of NADP added. When light was replaced by dithiotreitol, the inhibition effect was even more pronounced. It was assumed that NADP inhibits the modulation reaction directly: reduced thioredoxin, a potent mediator of activation by light, or dithiotreitol appear to counteract NADP in a competitive manner. The results indicate a physiological role of NADP in the regulation of chloroplastic NADP-dependent malate dehydrogenase which is capable of removing electrons from the chloroplast, via oxaloacetate reduction and malate export. Thus an NADP concentration sufficient for continuous photosynthetic electron flow may be achieved.     

Regulation of C4 photosynthesis: Physical and kinetic properties of active (dithiol) and inactive (disulfide) NADP-malate dehydrogenase from Zea mays

NADP-malate dehydrogenase was purified from leaves of Zea mays in the absence of thiol-reducing agents by (NH₄)₂SO₄, polyethylene glycol, and pH fractionation followed by dye-ligand affinity chromatography and gel filtration. The purified enzyme is completely inactive (no activity detected between pH 6 and 9) but can be reactivated by thiol-reducing agents including dithiothreitol and thioredoxin. The active enzyme shows distinctly alkaline pH optima when assayed in either direction; K_m values at pH 8.5 are oxaloacetate, 18 μm; malate, 24 mm; NADPH, 50 μm; and NADP, 45 μm. The reduction of oxaloacetate is inhibited by NADP (competitive with respect to NADPH, K_i = 50 μm). The molecular weight of the native inactive or active enzyme is 150,000 with subunits of M_r 38,000. Active enzyme is much more sensitive (>50-fold) to heat denaturation than is the inactive enzyme and is irreversibly inactivated by N-ethylmaleimide whereas the inactive enzyme is insensitive to this reagent. The active and inactive forms of NADP-malate dehydrogenase are assumed to correspond to dithiol and disulfide forms of the enzyme, respectively. The relative coenzyme-binding affinities of inactive NADP-malate dehydrogenase differ by a factor of 10² from the binding affinities for active NADP-malate dehydrogenase and 10⁴ for non-thiol-regulated NAD-specific malate dehydrogenase. It is proposed that the 100-fold change in differential binding of NADP and NADPH upon conversion of NADP-malate dehydrogenase to the disulfide form may sufficiently alter the equilibrium of the central enzyme-substrate complexes, and hence the catalytic efficiency of the enzyme, to explain the associated loss of activity.     

光还原的硫氧还蛋白对6—磷酸葡萄糖脱氢酶的钝化作用

测定了豌豆(Pisum sativum)幼苗的重组叶绿体中光还原的硫氧还蛋白(Td)对6-磷酸葡萄糖脱氢酶(G6PDH)的钝化作用.结果表明,Td在叶绿体G6PDH的光抑制和暗激活中均起重要的调节作用.在其绿色叶片和黄化组织中,G6PDH都存在着两种同工酶,但二硫苏糖醇(DTT)和Td对黄化幼苗中G6PDH活性的影响与叶绿体的明显不同,DTT对黄化幼苗G6PDH的钝化作用和氧化Td的活化作用均低于对叶绿体中的这两种作用.     

Expression and characterization of pea chloroplastic glyceraldehyde-3-phosphate dehydrogenase composed of only the B-subunit.

A cDNA fragment coding for the pea (Pisum sativum L.) chloroplastic glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) B-subunit and a truncated form corresponding in length to the A-subunit have been cloned into an expression vector, expressed in the absence of the A-subunit in a gap- Escherichia coli strain, purified, and studied. Like the isolated enzyme from higher plant chloroplasts, the recombinant enzymes have dual specificity for NADPH and NADH. The recombinant glyceraldehyde-3-P dehydrogenases have the same optimal pH as the enzyme isolated from pea chloroplasts. Like the native chloroplast enzyme, the recombinant B-subunit has a marked tendency to form large aggregates, whereas the truncated B-subunit exists as the tetramer. The recombinant B-subunit glyceraldehyde 3-P dehydrogenase is more sensitive to dithiothreitol than its truncated form. It seems likely that a different pair of cysteines is responsible for the redox sensitivity of the activity of the enzyme composed of B-subunits than the cysteine residues implicated in the modulation of the activity of the enzyme composed of A-subunits by previous work in this laboratory.     

Light activation of nadp malate dehydrogenase in a reconstituted chloroplastic system

Isolated spinach chloroplasts exhibited a light dependent NADP-malate dehydrogenase activity. In a reconstituted chloroplastic system, it was demonstrated that the activation process requires light, chloroplastic membranes, ferredoxin, protein factors and a component which is probably a reductase. Moreover, it was shown that activation of malate dehydrogenase by dithiothreitol or by light resulted in sulfhydryl groups formation on protein factors and enzyme. Using this system, activation of the bean leaf enzyme in the presence of its own protein factors was also obtained. The influence of the different components of the system on the activation process was investigated.     

Properties and regulation of leaf nicotinamide–adenine dinucleotide phosphate–malate dehydrogenase and `malic'' enzyme in plants with the C4-dicarboxylic acid pathway of photosynthesis

1. NADP-malate dehydrogenase and ;malic' enzyme in maize leaf extracts were separated from NAD-malate dehydrogenase and their properties were examined. 2. The NADP-malate dehydrogenase was nicotinamide nucleotide-specific but otherwise catalysed a reaction comparable with that with the NAD-specific enzyme. By contrast with the latter enzyme, a thiol was absolutely essential for maintaining the activity of the NADP-malate dehydrogenase, and the initial velocity in the direction of malate formation, relative to the reverse direction, was faster. 3. For the ;malic' enzyme reaction the K(m) for malate was dependent on pH and the pH optimum varied with the malate concentration. At their respective optimum concentrations the maximum velocity for this enzyme was higher with Mg(2+) than with Mn(2+). 4. The NADP-malate dehydrogenase in green leaves was rapidly inactivated in the dark and was reactivated when plants were illuminated. Reactivation of the enzyme extracted from darkened leaves was achieved simply by adding a thiol compound. 5. The activity of both enzymes was low in etiolated leaves of maize plants grown in the dark but increased 10-20-fold, together with chlorophyll, when leaves were illuminated. 6. The activity of these enzymes in different species with the C(4)-dicarboxylic acid pathway was compared and their possible role in photosynthesis was considered.     

Deactivation of Glucose-6-Phosphate Dehydrogenase by Light-Reduced Thioredoxin

The activity of glucose-6-phosphate dehydrogenase (G6PDH, E. C. 1.1.1.49) in a reconsituted pea chloroplast system was assayed spectrophotometrically by the reduction of NADP, ming glucose-6-phosphate as substrate. Deactivation of G6PDH could be intensified by adding lightreduced thioredoxin (Td) into the reconstituted chloroplast system. The experimental results presented suggest that Td plays an important role not only in the dark activation, but also in the light deactivation of G6PDH in chloroplasts. There were two isozymes of G6PDH in green and in etiolated pea seedlings. The effects of dithiothreitol (DTT) and Td on G6PDH in etiolated seedlings were different from that in chloroplasts. The light regulation of G6PDH in chloroplasts is mediated through Td.     

Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts

Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.     

Primary structure and analysis of the location of the regulatory disulfide bond of pea chloroplast NADP-malate dehydrogenase

Purified pea chloroplast NADP-malate dehydrogenase (S)-malate: NADP⁺ oxidoreductase, EC 1.1.1.82) was digested with trypsin and the resulting peptides were separated by HPLC and sequenced. Together with the information from earlier work (Fickenscher, K. et al. (1987) Eur. J. Biochem. 168, 653–658) the total sequence is now known to an extent of 78%. Comparison with the sequence of the corn NADP-malate dehydrogenase deduced from its cDNA (Metzler, M.C. et al. (1989) Plant Mol. Biol. 12, 713–722) showed 84% agreement; however, the 11 N-terminal residues exhibit only 27% similarity. The N- and C-terminal extrapeptides of the pea NADP-malate dehydrogenase when aligned with non-regulatory NAD-malate dehydrogenases from bacteria or mammals consist of 30 and 17 amino acids, respectively. Since all cysteine-containing peptides were sequenced, the number of eight cysteines per subunit of the pea enzyme was established. The native, oxidized enzyme ss characterized by an extremely slow reactivity of two thiols. Titration of the thiols of the denatured, oxidized enzyme both with DTNB and with pCMB resulted in six thiols not involved in disulfide formation. Therefore, one disulfide bridge must be present per 38.9 kDa subunit. Analysis of disulfide bonds by urea gel electrophoresis confirmed this finding. Using digestion products of NADP-malate dehydrogenase with aminopeptidase K, the location of the single disulfide bridge was established to be on the N-terminal arm (Cys-12 and Cys-17) of the polypeptide chain.