Developmental physiology of cluster-root carboxylate synthesis and exudation in harsh hakea. Expression of phosphoenolpyruvate carboxylase and the alternative oxidase.
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Harsh hakea (Hakea prostrata R.Br.) is a member of the Proteaceae family, which is highly represented on the extremely nutrient-impoverished soils in southwest Australia. When phosphorus is limiting, harsh hakea develops proteoid or cluster roots that release carboxylates that mobilize sparingly soluble phosphate in the rhizosphere. To investigate the physiology underlying the synthesis and exudation of carboxylates from cluster roots in Proteaceae, we measured O2 consumption, CO2 release, internal carboxylate concentrations and carboxylate exudation, and the abundance of the enzymes phosphoenolpyruvate carboxylase and alternative oxidase (AOX) over a 3-week time course of cluster-root development. Peak rates of citrate and malate exudation were observed from 12- to 13-d-old cluster roots, preceded by a reduction in cluster-root total protein levels and a reduced rate of O2 consumption. In harsh hakea, phosphoenolpyruvate carboxylase expression was relatively constant in cluster roots, regardless of developmental stage. During cluster-root maturation, however, the expression of AOX protein increased prior to the time when citrate and malate exudation peaked. This increase in AOX protein levels is presumably needed to allow a greater flow of electrons through the mitochondrial electron transport chain in the absence of rapid ATP turnover. Citrate and isocitrate synthesis and accumulation contributed in a major way to the subsequent burst of citrate and malate exudation. Phosphorus accumulated by harsh hakea cluster roots was remobilized during senescence as part of their efficient P cycling strategy for growth on nutrient impoverished soils. (+info)
Site-directed mutagenesis of the phosphorylatable serine (Ser8) in C4 phosphoenolpyruvate carboxylase from sorghum. The effect of negative charge at position 8.
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The properties of the dephospho and in vitro phosphorylated forms of recombinant sorghum phosphoenolpyruvate carboxylase have been compared with those of the authentic dark (dephospho) and light (phospho) leaf enzyme forms and two mutant enzymes in which the phosphorylatable serine residue (Ser8) has been changed by site-directed mutagenesis to Cys (S8C) or Asp (S8D). Kinetic analysis of the purified recombinant, mutant, and leaf enzyme forms at pH 8.0 indicated virtually identical Vmax, apparent Km (phosphoenolpyruvate), and half-maximal activation (glucose 6-P) values of about 44 units/mg, 1.1 mM, and 0.23 mM, respectively. In contrast, the Ser8, S8C, and dark leaf enzymes were about 3-fold more sensitive to inhibition by L-malate at pH 7.3 than the Ser8-P, S8D, and light leaf enzyme forms. These comparative results indicate that: (i) Ser8 is an important determinant in the regulation of sorghum phosphoenolpyruvate carboxylase activity by negative (L-malate), but not positive (glucose 6-phosphate) metabolite effectors, (ii) phosphorylation of this target residue can be functionally mimicked by Asp, but not Cys, and (iii) negative charge contributes to the effect of regulatory phosphorylation on this C4-photosynthesis enzyme. (+info)
Expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxylase kinase genes. Implications for genotypic capacity and phenotypic plasticity in the expression of crassulacean acid metabolism.
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In plants with crassulacean acid metabolism (CAM), dark CO2 uptake is mediated by phosphoenolpyruvate carboxylase (PEPC), an enzyme that can be regulated at transcriptional and posttranslational levels. Reversible phosphorylation of PEPC is catalyzed by a dedicated PEPC kinase, which in turn is regulated at the transcriptional level over the 24-h cycle in CAM plants. PEPC kinase controls the day/night regulation of PEPC during the CAM cycle, thus facilitating plasticity for optimizing CO2 uptake under different environmental conditions. To understand the importance of PEPC kinase in relation to its target PEPC in terms of CAM performance, the expression of the genes encoding the two enzymes was investigated in four species of Clusia that have photosynthetic patterns ranging from C3 photosynthesis to constitutive CAM. By linking changes in the expression of PEPC and PEPC kinase to day/night patterns of leaf gas exchange, organic acid, and soluble sugar contents under different environmental conditions, the genetic and metabolic limitations to CAM plasticity were assessed. The results indicate that PEPC expression is a major factor underpinning the genotypic capacity for CAM and that PEPC kinase expression does not appear to limit CAM. The day/night regulation of Ppck transcript abundance was found to be a consequence of CAM and the day/night cycling of associated metabolites, rather than the primary controlling factor for the temporal separation of carboxylation processes. (+info)
Metabolic engineering with Dof1 transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions.
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Utilization of transcription factors might be a powerful approach to modification of metabolism for a generation of crops having superior characteristics because a single transcription factor frequently regulates coordinated expression of a set of key genes for respective pathways. Here, we apply the plant-specific Dof1 transcription factor to improve nitrogen assimilation, the essential metabolism including the primary assimilation of ammonia to carbon skeletons to biosynthesize amino acids and other organic compounds involving nitrogen in plants. Expressing Dof1 induced the up-regulation of genes encoding enzymes for carbon skeleton production, a marked increase of amino acid contents, and a reduction of the glucose level in transgenic Arabidopsis. The results suggest cooperative modification of carbon and nitrogen metabolisms on the basis of their intimate link. Furthermore, elementary analysis revealed that the nitrogen content increased in the Dof1 transgenic plants (approximately 30%), indicating promotion of net nitrogen assimilation. Most significantly, the Dof1 transgenic plants exhibit improved growth under low-nitrogen conditions, an agronomically important trait. These results highlight the great utility of transcription factors in engineering metabolism in plants. (+info)
The phosphoenolpyruvate carboxylase from Methanothermobacter thermautotrophicus has a novel structure.
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In Methanothermobacter thermautotrophicus, oxaloacetate synthesis is a major and essential CO(2)-fixation reaction. This methanogenic archaeon possesses two oxaloacetate-synthesizing enzymes, pyruvate carboxylase and phosphoenolpyruvate carboxylase. The phosphoenolpyruvate carboxylase from this organism was purified to homogeneity. The subunit size of this homotetrameric protein was 55 kDa, which is about half that of all known bacterial and eukaryotic phosphoenolpyruvate carboxylases (PPCs). The NH(2)-terminal sequence identified this enzyme as the product of MTH943, an open reading frame with no assigned function in the genome sequence. A BLAST search did not show an obvious sequence similarity between MTH943 and known PPCs, which are generally well conserved. This is the first report of a new type of phosphoenolpyruvate carboxylase that we call PpcA ("A" for "archaeal"). Homologs to PpcA were present in most archaeal genomic sequences, but only in three bacterial (Clostridium perfringens, Oenococcus oeni, and Leuconostoc mesenteroides) and no eukaryotic genomes. PpcA was the only recognizable oxaloacetate-producing enzyme in Methanopyrus kandleri, a hydrothermal vent organism. Each PpcA-containing organism lacked a PPC homolog. The activity of M. thermautotrophicus PpcA was not influenced by acetyl coenzyme A and was about 50 times less sensitive to aspartate than the Escherichia coli PPC. The catalytic core (including His(138), Arg(587), and Gly(883)) of the E. coli PPC was partly conserved in PpcA, but three of four aspartate-binding residues (Lys(773), Arg(832), and Asn(881)) were not. PPCs probably evolved from PpcA through a process that added allosteric sites to the enzyme. The reverse is also equally possible. (+info)
Phosphate deficiency regulates phosphoenolpyruvate carboxylase expression in proteoid root clusters of white lupin.
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Proteoid roots play a major role in enabling white lupin (Lupinus albus L.) to adapt to phosphate (Pi) deficiency. Such roots release citrate from proteoid rootlets, which allows this species to mobilize Pi from sparingly soluble Pi sources. Release of citrate is preceded by a significant accumulation of organic acids, in which a Pi deficiency-inducible phosphoenolpyruvate carboxylase (PEPC) activity has been involved. To gain an insight into this adaptive mechanism, the expression of three different transcripts coding for PEPC was examined in proteoid rootlets of Pi-starved and Pi-starved-and-rescued white lupin. Semi-quantitative reverse transcriptase (RT)-PCR experiments performed with gene-specific primers targeted to the 3'-end region of the corresponding cDNAs revealed that the transcripts for these three PEPCs differentially accumulate in both Pi-starved and Pi-starved-and-rescued proteoid rootlets. Semi-quantitative RT-PCR analysis in Pi-starved proteoid rootlets sampled at different times after being rescued from Pi deficiency showed that Pi levels differentially down-regulated the three PEPC transcripts. RT-PCR experiments were further extended to Pi-starved and Pi-fed whole roots, cotyledons, and leaves on which a tissue-specific, Pi-dependent PEPC expression was observed. These results indicate that there exists at least three different transcripts coding for PEPC in proteoid root clusters of white lupin, whose expression are differentially regulated by Pi. (+info)
Identification and functional verification of archaeal-type phosphoenolpyruvate carboxylase, a missing link in archaeal central carbohydrate metabolism.
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Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some Archaea, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized archaeal gene family that is distantly related to the catalytic core of the canonical PEPC. To verify the predicted function of this archaeal gene family, we cloned a representative from the hyperthermophilic acidophile Sulfolobus solfataricus and functionally produced the corresponding enzyme as a fusion with the Escherichia coli maltose-binding protein. The purified fusion protein indeed displayed highly thermostable PEPC activity. The structural and biochemical properties of the characterized archaeal-type PEPC (atPEPC) from S. solfataricus are in good agreement with previously reported biochemical analyses of other archaeal PEPC enzymes. The newly identified atPEPC, with its distinct properties, constitutes yet another example of the versatility of the enzymes of the central carbon metabolic pathways in the archaeal domain. (+info)
Maize phosphoenolpyruvate carboxylase. Mutations at the putative binding site for glucose 6-phosphate caused desensitization and abolished responsiveness to regulatory phosphorylation.
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Phosphoenolpyruvate carboxylases (PEPC, EC 4.1.1.31) from higher plants are regulated by both allosteric effects and reversible phosphorylation. Previous x-ray crystallographic analysis of Zea mays PEPC has revealed a binding site for sulfate ion, speculated to be the site for an allosteric activator, glucose 6-phosphate (Glc-6-P) (Matsumura, H., Xie, Y., Shirakata, S., Inoue, T., Yoshinaga, T., Ueno, Y., Izui, K., and Kai, Y. (2002) Structure (Lond.) 10, 1721-1730). Because kinetic experiments have also supported this notion, each of the four basic residues (Arg-183, -184, -231, and -372' on the adjacent subunit) located at or near the binding site was replaced by Gln, and the kinetic properties of recombinant mutant enzymes were investigated. Complete desensitization to Glc-6-P was observed for R183Q, R184Q, R183Q/R184Q (double mutant), and R372Q, as was a marked decrease in the sensitivity for R231Q. The heterotropic effect of Glc-6-P on an allosteric inhibitor, l-malate, was also abolished, but sensitivity to Gly, another allosteric activator of monocot PEPC, was essentially not affected, suggesting the distinctness of their binding sites. Considering the kinetic and structural data, Arg-183 and Arg-231 were suggested to be involved directly in the binding with phosphate group of Glc-6-P, and the residues Arg-184 and Arg-372 were thought to be involved in making up the site for Glc-6-P and/or in the transmission of an allosteric regulatory signal. Most unexpectedly, the mutant enzymes had almost lost responsiveness to regulatory phosphorylation at Ser-15. An apparent lack of kinetic competition between the phosphate groups of Glc-6-P and of phospho-Ser at 15 suggested the distinctness of their binding sites. The possible roles of these Arg residues are discussed. (+info)