Screening Marine Fungi for Inhibitors of the C4 Plant Enzyme Pyruvate Phosphate Dikinase: Unguinol as a Potential Novel Herbicide Candidate

A total of 2,245 extracts, derived from 449 marine fungi cultivated in five types of media, were screened against the C₄ plant enzyme pyruvate phosphate dikinase (PPDK), a potential herbicide target. Extracts from several fungal isolates selectively inhibited PPDK. Bioassay-guided fractionation of one isolate led to the isolation of the known compound unguinol, which inhibited PPDK with a 50% inhibitory concentration of 42.3 ± 0.8 μM. Further kinetic analysis revealed that unguinol was a mixed noncompetitive inhibitor of PPDK with respect to the substrates pyruvate and ATP and an uncompetitive inhibitor of PPDK with respect to phosphate. Unguinol had deleterious effects on a model C₄ plant but no effect on a model C₃ plant. These results indicate that unguinol inhibits PPDK via a novel mechanism of action which also translates to an herbicidal effect on whole plants.     

Primary structure of pyruvate, orthophosphate dikinase in the dicotyledonous C4 plant Flaveria trinervia

We have isolated and characterized cDNA clones encoding the entire precursor for the leafspecific isoform of pyruvate, orthophosphate dikinase (PPDK) from the dicotyledonous C4 plant Flaveria trinervia. The deduced amino acid sequence reveals a high degree of similarity to the corresponding maize protein indicating a common evolutionary basis. However, no significant similarities are apparent upon comparison of the putative transit peptides. The implications of this divergence are discussed with respect to the evolution of PPDK genes.     

植物丙酮酸磷酸双激酶研究进展

丙酮酸磷酸双激酶(pyruvate orthophosphate dikinase,PPDK)作为C4光合途径中一个非常重要的限速酶,其功能已经清楚,但在C3植物中以及逆境条件下的作用尚不明确。在阐述PPDK基本生物学特征的基础上,重点介绍了PPDK在C4植物和C3植物中的功能、活性调控、基因工程以及PPDK对逆境胁迫应答的研究进展,以期为植物抗逆基因挖掘及抗逆种质创制提供参考。     

Crystal structures of pyruvate phosphate dikinase from maize revealed an alternative conformation in the swiveling-domain motion

Pyruvate phosphate dikinase (PPDK) reversibly catalyzes the conversion of ATP, phosphate, and pyruvate into AMP, pyrophosphate, and phosphoenolpyruvate (PEP), respectively. Since the nucleotide binding site (in the N-terminal domain) and the pyruvate/PEP binding site (in the C-terminal domain) are separated by approximately 45 A, it has been proposed that an intermediary domain, called the central domain, swivels between these remote domains to transfer the phosphate. However, no direct structural evidence for the swiveling central domain has been found. In this study, the crystal structures of maize PPDK with and without PEP have been determined at 2.3 A resolution. These structures revealed that the central domain is located near the pyruvate/PEP binding C-terminal domain, in contrast to the PPDK from Clostridium symbiosum, wherein the central domain is located near the nucleotide-binding N-terminal domain. Structural comparisons between the maize and C. symbiosum PPDKs demonstrated that the swiveling motion of the central domain consists of a rotation of at least 92 degrees and a translation of 0.5 A. By comparing the maize PPDK structures with and without PEP, we have elucidated the mode of binding of PEP to the C-terminal domain and the induced conformational changes in the central domain.     

ATP generation in the Trypanosoma brucei procyclic form: cytosolic substrate level is essential, but not oxidative phosphorylation

Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic form of this parasite, transmitted by tsetse flies, is considered to be dependent on oxidative phosphorylation for ATP production. Indeed, its respiration was 55% inhibited by oligomycin, which is the most specific inhibitor of the mitochondrial F0/F1-ATP synthase. However, a 10-fold excess of this compound did not significantly affect the intracellular ATP concentration and the doubling time of the parasite was only 1.5-fold increased, suggesting that oxidative phosphorylation is not essential for procyclic trypanosomes. To further investigate the sites of ATP production, we studied the role of two ATP producing enzymes, which are involved in the synthesis of pyruvate from phosphoenolpyruvate: the glycosomal pyruvate phosphate dikinase (PPDK) and the cytosolic pyruvate kinase (PYK). The parasite was not affected by PPDK gene knockout. In contrast, inhibition of PYK expression by RNA interference was lethal for these cells. In the absence of PYK activity, the intracellular ATP concentration was reduced by up to 2.3-fold, whereas the intracellular pyruvate concentration was not reduced. Furthermore, we show that this mutant cell line still excreted acetate from d-glucose metabolism, and both the wild type and mutant cell lines consumed pyruvate present in the growth medium with similar high rates, indicating that in the absence of PYK activity pyruvate is still present in the trypanosomes. We conclude that PYK is essential because of its ATP production, which implies that the cytosolic substrate level phosphorylation is essential for the growth of procyclic trypanosomes.     

Cytosolic pyruvate,orthophosphate dikinase functions in nitrogen remobilization during leaf senescence and limits individual seed growth and nitrogen content

The protein content of seeds determines their nutritive value, downstream processing properties and market value. Up to 95% of seed protein is derived from amino acids that are exported to the seed after degradation of existing protein in leaves, but the pathways responsible for this nitrogen metabolism are poorly defined. The enzyme pyruvate,orthophosphate dikinase (PPDK) interconverts pyruvate and phosphoenolpyruvate, and is found in both plastids and the cytosol in plants. PPDK plays a cardinal role in C₄ photosynthesis, but its role in the leaves of C₃ species has remained unclear. We demonstrate that both the cytosolic and chloroplastic isoforms of PPDK are up‐regulated in naturally senescing leaves. Cytosolic PPDK accumulates preferentially in the veins, while chloroplastic PPDK also accumulates in mesophyll cells. Analysis of microarrays and labelling patterns after feeding ¹³C‐labelled pyruvate indicated that PPDK functions in a pathway that generates the transport amino acid glutamine, which is then loaded into the phloem. In Arabidopsis thaliana, over‐expression of PPDK during senescence can significantly accelerate nitrogen remobilization from leaves, and thereby increase rosette growth rate and the weight and nitrogen content of seeds. This indicates an important role for cytosolic PPDK in the leaves of C₃ plants, and allows us to propose a metabolic pathway that is responsible for production of transport amino acids during natural leaf senescence. Given that increased seed size and nitrogen content are desirable agronomic traits, and that efficient remobilization of nitrogen within the plant reduces the demand for fertiliser applications, PPDK and the pathway in which it operates are targets for crop improvement.     

A rapid screening method to detect specific inhibitors of pyruvate orthophosphate dikinase as leads for C4 plant-selective herbicides

Plants using the C(4) photosynthetic pathway are highly represented among the world's worst weeds, with only 4 C(4) species being agriculturally productive (maize, sorghum, millet, and sugar cane). With the C(4) acid cycle operating as a biochemical appendage of C(3) photosynthesis, the additional enzymes involved in C(4) photosynthesis represent an attractive target for the development of weed-specific herbicides. The rate-limiting enzyme of this metabolic pathway is pyruvate orthophosphate dikinase (PPDK). PPDK, coupled with phosphoenolpyruvate carboxylase and nicotinamide adenine dinucleotide-malate dehydrogenase, was used to develop a microplate-based assay to detect inhibitors of enzymes of the C(4) acid cycle. The resulting assay had a Z' factor of 0.61, making it a high-quality assay able to reliably identify active test samples. Organic extracts of 6679 marine macroscopic organisms were tested within the assay, and 343 were identified that inhibited the 3 enzyme-coupled reaction. A high confirmation rate was achieved, with 95% of these hit extracts proving active again upon retesting. Sequential addition of phosphoenolpyruvate and oxaloacetate to the assay facilitated identification of 83 extracts that specifically inhibited PPDK.     

The role of inorganic phosphate in the regulation of C4 photosynthesis

Inorganic phosphate participates in many fundamental processes within the plant cell. Its broad influence on plant metabolism is related to such key operations as metabolite transport, enzyme regulation and carbohydrate metabolism in general. This review discusses these topics with special emphasis on the role assigned to this ubiquitous anion within the C₄ pathway of photosynthesis.Abbreviations DHAP dihydroxyacetone phosphate - Ga3P glyceraldehyde-3-phosphate - NAD(P)-ME-NAD(P) dependent malic enzyme - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - PFK and PFP-ATP- and PPi dependent fructose-6-phosphate 1-phosphotransferase - PPDK pyruvate:orthophosphate dikinase - RPPC reductive pentose-phosphate cycle - RuBisCO ribulose bisphosphate carboxylase-oxygenase - SPS sucrose-6-phosphate synthase     

Examination of the structure, stability, and catalytic potential in the engineered phosphoryl carrier domain of pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) is a multidomain protein that catalyzes the interconversion of ATP, pyruvate, and phosphate with AMP, phosphoenolpyruvate (PEP), and pyrophosphate using its central domain to transport phosphoryl groups between two distant active sites. In this study, the mechanism by which the central domain moves between the two catalytic sites located on the N-terminal and C-terminal domains was probed by expressing this domain as an independent protein and measuring its structure, stability, and ability to catalyze the ATP/phosphate partial reaction in conjunction with the engineered N-terminal domain protein (residues 1-340 of the native PPDK). The encoding gene was engineered to express the central domain as residues 381-512 of the native PPDK. The central domain was purified and shown to be soluble, monomeric (13,438 Da), and stable (deltaG = 4.3 kcal/mol for unfolding in buffer at pH 7.0, 25 degrees C) and to possess native structure, as determined by multidimensional heteronuclear NMR analysis. The main chain structure of the central domain in solution aligns closely with that of the X-ray structure of native PPDK (the root-mean-square deviation is 2.2 A). Single turnover reactions of [14C]ATP and phosphate, carried out in the presence of equal concentrations of central domain and the N-terminal domain protein, did not produce the expected products, in contrast to efficient product formation observed for the N-terminal central domain construct (residues 1-553 of the native PPDK). These results are interpreted as evidence that the central domain, although solvent-compatible, must be tethered by the flexible linkers to the N-terminal domain for the productive domain-domain docking required for efficient catalysis.     

Adaptation responses in C4 photosynthesis of maize under salinity

The effect of salinity on C(4) photosynthesis was examined in leaves of maize, a NADP-malic enzyme (NADP-ME) type C(4) species. Potted plants with the fourth leaf blade fully developed were treated with 3% NaCl solution for 5d. Under salt treatment, the activities of pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPCase), NADP-dependent malate dehydrogenase (NADP-MDH) and NAD-dependent malate dehydrogenase (NAD-MDH), which are derived mainly from mesophyll cells, increased, whereas those of NADP-ME and ribulose-1,5-bisphosphate carboxylase, which are derived mainly from bundle sheath cells (BSCs), decreased. Immunocytochemical studies by electron microscopy revealed that PPDK protein increased, while the content of ribulose-1,5-bisphosphate carboxylase/oxygenase protein decreased under salinity. In salt-treated plants, the photosynthetic metabolites malate, pyruvate and starch decreased by 40, 89 and 81%, respectively. Gas-exchange analysis revealed that the net photosynthetic rate, the transpiration rate, stomatal conductance (g(s)) and the intercellular CO(2) concentration decreased strongly in salt-treated plants. The carbon isotope ratio (δ(13)C) in these plants was significantly lower than that in control. These findings suggest that the decrease in photosynthetic metabolites under salinity was induced by a reduction in gas-exchange. Moreover, in addition to the decrease in g(s), the decrease in enzyme activities in BSCs was responsible for the decline of C(4) photosynthesis. The increase of PPDK, PEPCase, NADP-MDH, and NAD-MDH activities and the decrease of NADP-ME activity are interpreted as adaptation responses to salinity.     

Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase

The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [alpha beta-18O, beta, beta-18O2]ATP and [beta gamma-18O, gamma, gamma, gamma-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light Activation of Pyruvate,Pi Dikinase and NADP-Malate Dehydrogenase in Mesophyll Protoplasts of Maize : Effect of DCMU, Antimycin A, CCCP, and Phlorizin

Pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) and NADP-malate dehydrogenase (MDH, EC 1.1.1.82) were activated in the light and inactivated following a dark treatment in mesophyll protoplasts of maize. DCMU (up to 33 micromolar), an inhibitor of noncyclic electron transport, inhibited activation of MDH much more strongly than it did PPDK. Antimycin A (6.6-33 micromolar), an inhibitor of cyclic photophosphorylation, inhibited the activation of PPDK (up to 61%), but had little or no effect on activation of MDH. Carbonyl cyanide m-chlorophenylhydrazone (0.2-2 micromolar) and nigericin (0.4 micromolar), uncouplers of photophosphorylation, inhibited activation of PPDK while stimulating the activation of MDH. Phlorizin (0.33-1.7 millimolar), an inhibitor of the coupling factor for ATP synthesis, strongly inhibited activation of PPDK but only slightly effected light activation of MDH. These results suggest that noncyclic electron flow is required for activation of NADP-MDH and that photophosphorylation is required for activation of PPDK.     

Studies on the dark/light regulation of maize leaf pyruvate, orthophosphate dikinase by reversible phosphorylation

In maize leaves, pyruvate, orthophosphate dikinase (PPDK) is deactivated in the dark and reactivated in the light. Studies in vitro using purified PPDK and a partially purified regulatory protein from maize confirmed previous reports correlating deactivation/reactivation with the reversible phosphorylation/dephosphorylation of a threonyl residue. By monitoring the stability of the exogenous 32P-labeled adenylate substrates during deactivation, we have firmly established ADP as the specific phosphate donor. In isolated maize leaf mesophyll protoplasts preilluminated with 32Pi, we observed a three- to fivefold higher PPDK activity in situ in the light, and a corresponding three- to fivefold higher level of phosphorylation of the 94-kDa PPDK protomer in the dark. HPLC-based phosphoamino acid analysis of PPDK purified from maize leaves of both light- and dark-adapted plants revealed the presence of P-serine. The inactive enzyme from dark-adapted plants (inactivated in vivo) also contained P-threonine. Total phosphate content of PPDK purified from leaves of light-adapted plants was approximately 0.5 mol/mol protomer, and 1.5 mol/mol protomer from leaves of dark-adapted plants. Since the difference between enzyme purified from light-adapted (active PPDK) and dark-adapted (inactive PPDK) plants is the presence of P-threonine in the latter, this suggests an inactivation stoichiometry in vivo of 1 mol P-threonine/mol 94-kDa protomer. These complementary studies with maize leaf PPDK in vitro, in situ, and in vivo provide convincing evidence for the dark/light regulation of this key C4-photosynthesis enzyme by reversible phosphorylation.     

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

The role of pyruvate metabolism in the triggering of aerobic, alcoholic fermentation in Saccharomyces cerevisiae has been studied. Since Candida utilis does not exhibit a Crabtree effect. this yeast was used as a reference organism. The localization, activity and kinetic properties of pyruvate carboxylase (EC 6.4.1.1), the pyruvate dehydrogenase complex and pyruvate decarboxylase (EC 4.1.1.1) in cells of glucose-limited chemostat cultures of the two yeasts were compared. In contrast to the general situation in fungi, plants and animals, pyruvate carboxylase was found to be a cytosolic enzyme in both yeasts. This implies that for anabolic processes, transport of C4-dicarboxylic acids into the mitochondria is required. Isolated mitochondria from both yeasts exhibited the same kinetics with respect to oxidation of malate. Also, the affinity of isolated mitochondria for pyruvate oxidation and the in situ activity of the pyruvate dehydrogenase complex was similar in both types of mitochondria. The activity of the cytosolic enzyme pyruvate decarboxylase in S. cerevisiae from glucose-limited chemostat cultures was 8-fold that in C. utilis. The enzyme was purified from both organisms, and its kinetic properties were determined. Pyruvate decarboxylase of both yeasts was competitively inhibited by inorganic phosphate. The enzyme of S. cerevisiae was more sensitive to this inhibitor than the enzyme of C. utilis. The in vivo role of phosphate inhibition of pyruvate decarboxylase upon transition of cells from glucose limitation to glucose excess and the associated triggering of alcoholic fermentation was investigated with 31P-NMR. In both yeasts this transition resulted in a rapid drop of the cytosolic inorganic phosphate concentration. It is concluded that the relief from phosphate inhibition does stimulate alcoholic fermentation, but it is not a prerequisite for pyruvate decarboxylase to become active in vivo. Rather, a high glycolytic flux and a high level of this enzyme are decisive for the occurrence of alcoholic fermentation after transfer of cells from glucose limitation to glucose excess.     

Dihydrodipicolinate synthase (DHDPS) from Escherichia coli displays partial mixed inhibition with respect to its first substrate, pyruvate

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) mediates the first unique reaction of (S)-lysine biosynthesis in plants and microbes-the condensation of (S)-aspartate-beta-semialdehyde ((S)-ASA) and pyruvate. It has been shown that DHDPS is partially feedback inhibited by (S)-lysine; it is suggested that this mechanism regulates flux through the DAP biosynthetic pathway. Others have characterised DHDPS from Escherichia coli with respect to (S)-lysine inhibition. They have concluded that, with respect to pyruvate, the first substrate of the reaction, DHDPS shows uncompetitive inhibition: as such, they further suggest that (S)-lysine inhibits DHDPS via interaction with the binding site for the second substrate, (S)-ASA. Yet, this finding is based on the assumption that (S)-lysine is a fully uncompetitive inhibitor. In light of crystallographic studies, which lead to the proposal that (S)-lysine affects the putative proton-relay of DHDPS, we re-evaluated the inhibition mechanism of DHDPS with respect to (S)-lysine by incorporating the observed hyperbolic inhibition. Our data showed that lysine is not an uncompetitive inhibitor, but a mixed inhibitor when pyruvate and (S)-lysine concentrations were varied. Thus, consistent with the crystallographic data, (S)-lysine must have an effect on the initial steps of the DHDPS reaction, including the binding of pyruvate and Schiff base formation.     

Cold stability of pyruvate,orthophosphate dikinase of Flaveria brownii

The nucleotide sequences of the complementary DNA of pyruvate, P_i dikinase (PPDK) from Flaveria bidentis, a C₄ plant which possesses a cold-sensitive form of PPDK, and Flaveria brownii, a C₄-like plant which possesses a cold-tolerant form of PPDK, were determined. PPDK was isolated from the leaves of both Flaveria species and purified and the N-terminal amino acid sequences characterised. Together with a maize PPDK cDNA, cDNA inserts which code for the mature form of PPDK of F. bidentis and of F. brownii were expressed in bacteria and the cold sensitivity of the expressed PPDK studied. The cold sensitivity of the PPDK expressed in bacteria mimics the cold sensitivity of PPDK found in vivo in all three plant species. This study indicates that the cold sensitivity of plant PPDK is controlled by the primary structure of the enzyme.