Role of NADP+ (corrected)-linked malic enzymes as regulators of the pool size of tricarboxylic acid-cycle intermediates in the perfused rat heart.
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ABSTRACT:
SUBMITTER: Sundqvist KE
PROVIDER: S-EPMC1147935 | biostudies-other | 1987 May
REPOSITORIES: biostudies-other
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Project description:BackgroundDespite significant limitations of growth medium reuse, a large amount of organic substrate is reused in soilless cultivation of horticultural crops in China. Arbuscular mycorrhizal fungi (AMF) can promote nutrient absorption and improve plant tolerance to biotic and abiotic stresses. However, the mechanisms governing the effects of AMF on crop growth in organic continuous cropping substrates have not been elucidated.ResultsIn this study, we showed that the inoculation of AMF in continuous cropping substrates promoted growth and root development, and increased the root and NADP-malic enzyme (NADP-ME) activity of tomato seedlings. Root transcriptome analysis demonstrated that the plant hormone signal transduction pathway was highly enriched, and 109 genes that positively correlated with the AMF-inoculated plant phenotype were obtained by gene set enrichment analysis (GSEA), which identified 9 genes related to indole acetic acid (IAA). Importantly, the levels of endogenous IAA in tomato seedlings significantly increased after AMF inoculation. Furthermore, the application of AMF significantly increased the expression levels of NADP-ME1 and NADP-ME2, as well as the activity of NADP-ME, and enhanced the root activity of tomato seedlings in comparison to that observed without inoculation of AMF. However, these effects were blocked in plants treated with 2,3,5-triiodobenzoic acid (TIBA), a polar transport inhibitor of IAA.ConclusionsThese results suggest that IAA mediates the AMF-promoted tomato growth and expression of NADP-MEs in continuous cropping substrates. The study provides convincing evidence for the reuse of continuous cropping substrates by adding AMF as an amendment.
| S-EPMC7814736 | biostudies-literature
Project description:C(4)-specific (photosynthetic) NADP(+)-dependent malic enzyme (NADP(+)-ME) has evolved from C(3)-malic enzymes and represents a unique and specialized form, as indicated by its particular kinetic and regulatory properties. In the present paper, we have characterized maize (Zea mays L.) photosynthetic NADP(+)-ME mutants in which conserved basic residues (lysine and arginine) were changed by site-directed mutagenesis. Kinetic characterization and oxaloacetate partition ratio of the NADP(+)-ME K255I (Lys-255-->Ile) mutant suggest that the mutated lysine residue is implicated in catalysis and substrate binding. Moreover, this residue could be acting as a base, accepting a proton in the malate oxidation step. At the same time, further characterization of the NADP(+)-ME R237L mutant indicates that Arg-237 is also a candidate for such role. These results suggest that both residues may play 'back-up' roles as proton acceptors. On the other hand, Lys-435 and/or Lys-436 are implicated in the coenzyme specificity (NADP(+) versus NAD(+)) of maize NADP(+)-ME by interacting with the 2'-phosphate group of the ribose ring. This is indicated by both the catalytic efficiency with NADP(+) or NAD(+), as well as by the reciprocal inhibition constants of the competitive inhibitors 2'-AMP and 5'-AMP, obtained when comparing the double mutant K435/6L (Lys-435/436-->Ile) with wild-type NADP(+)-ME. The results obtained in the present work indicate that the role of basic residues in maize photosynthetic NADP(+)-ME differs significantly with respect to its role in non-plant MEs, for which crystal structures have been resolved. Such differences are discussed on the basis of a predicted three-dimensional model of the enzyme.
| S-EPMC1133980 | biostudies-literature
Project description:The increase in the metabolite pool size of the tricarboxylic acid cycle in the isolated perfused rat heart after a decrease in the ATP consumption by KCl-induced arrest was used to study the anaplerotic mechanisms. During net anaplerosis the label incorporation into the tricarboxylic acid-cycle intermediates from [1-14C]pyruvate increased and occurred mainly by pathways not involving prior release of the label to CO2. A method for determination of the specific radioactivity of mitochondrial pyruvate was devised, and the results corroborated the notion that tissue alanine can be used as an indicator of the specific radioactivity of intracellular pyruvate.
| S-EPMC1154006 | biostudies-other
Project description:Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO(2) in the presence of divalent cations (Mg(2+), Mn(2+)). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD(+) or NADP(+), but not both. Structural studies of the human mitochondrial NAD(+)-dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP(+)-dependent malic enzyme, in a closed form, in a quaternary complex with NADP(+), Mn(2+), and oxalate. This represents the first structural information on an NADP(+)-dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2'-phosphate group of the NADP(+) cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP(+) selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.
| S-EPMC2373443 | biostudies-other
Project description:Malic enzyme (ME) comprises a family of proteins with multiple isoforms located in different compartments of eukaryotic cells. In plants, cytosolic and plastidic enzymes share several characteristics such as NADP specificity (NADP-ME), oxaloacetate decarboxylase (OAD) activity, and homo-oligomeric assembly. However, mitochondrial counterparts are NAD-dependent proteins (mNAD-ME) lacking OAD activity, which can be structured as homo- and hetero-oligomers of two different subunits. In this study, we examined the molecular basis of these differences using multiple sequence analysis, structural modeling, and phylogenetic approaches. Plant mNAD-MEs show the lowest identity values when compared with other eukaryotic MEs with major differences including short amino acid insertions distributed throughout the primary sequence. Some residues in these exclusive segments are co-evolutionarily connected, suggesting that they could be important for enzymatic functionality. Phylogenetic analysis indicates that eukaryotes from different kingdoms used different strategies for acquiring the current set of NAD(P)-ME isoforms. In this sense, while the full gene family of vertebrates derives from the same ancestral gene, plant NADP-ME and NAD-ME isoforms have a distinct evolutionary history. Plant NADP-ME genes may have arisen from the ?-protobacterial-like mitochondrial ancestor, a characteristic shared with major eukaryotic taxa. On the other hand, plant mNAD-ME genes were probably gained through an independent process involving the Archaeplastida ancestor. Finally, several residue signatures unique to all plant mNAD-MEs could be identified, some of which might be functionally connected to their exclusive biochemical properties. In light of these results, molecular evolutionary scenarios for these widely distributed enzymes in plants are discussed.
| S-EPMC5958461 | biostudies-literature
Project description:Arabidopsis thaliana possesses three cytosolic (NADP-ME1-3) and one plastidic (NADP-ME4) NADP-dependent malic enzymes. NADP-ME2 and -ME4 show constitutive expression, in contrast to NADP-ME1 and -ME3, which are restricted to particular tissues. Here, we show that NADP-ME1 transcript and protein were almost undetectable during normal vegetative growth, but gradually increased and reached levels higher than those of the other isoforms in the latest stages of seed development. Accordingly, in knockout nadp-me1 mature seeds the total NADP-ME activity was significantly lower than in wild type mature seeds. The phenotypic analysis of nadp-me1 plants indicated alterations of seed viability and germination. Besides, the treatment with abscisic acid (ABA), NaCl and mannitol specifically induced the accumulation of NADP-ME1 in seedlings. In line with this, nadp-me1 plants show a weaker response of primary and lateral root length and stomatal opening to the presence of ABA. The results suggest that NADP-ME1 plays a specialized role, linked to ABA signaling during the seed development as well as in the response to water deficit stress.
| S-EPMC6232891 | biostudies-literature
Project description:The amount of pulmonary surfactant within type II cells and in the alveolar space, referred to as surfactant pool sizes, are tightly regulated. The molecular pathways that sense and regulate surfactant pool size within the alveolus have not been identified and constitute a fundamental knowledge gap in the field. Our data show that mice with a germline mutation in the orphan G-protein-coupled receptor, GPR116, have a 30-fold accumulation of surfactant phospholipids that causes respiratory distress in adult animals. This phenotype is associated with increased surfactant secretion and induction of the purinergic receptor P2RY2 in young animals, and lipid-laden macrophages and alveolar destruction in older animals. We further demonstrate that GPR116 mRNA expression is developmentally regulated in the murine lung with peak expression at birth when surfactant pool sizes are maximal. Within the lung, GPR116 protein expression is restricted to the apical plasma membrane of alveolar type I and type II epithelial cells. To better understand the roles and molecular mechanisms by which Gpr116 influences gene expression in lung, the effect of cell-selective deletion of Gpr116 (Gpr116D/D) on genome-wide mRNA expression profiles was determined in murine type II alveolar epithelial cells. Differentially expressed genes were identified from Affymetrix Murine GeneChips analysis and subjected to gene ontology classification promoter analysis, pathway mapping and literature mining.
2013-04-10 | E-GEOD-41417 | biostudies-arrayexpress
Project description:Bacterial hybrid malic enzymes (MaeB grouping, multidomain) catalyse the transformation of malate to pyruvate, and are a major contributor to cellular reducing power and carbon flux. Distinct from other malic enzyme subtypes, the hybrid enzymes are regulated by acetyl-CoA, a molecular indicator of the metabolic state of the cell. Here we solve the structure of a MaeB protein, which reveals hybrid enzymes use the appended phosphotransacetylase (PTA) domain to form a hexameric sensor that communicates acetyl-CoA occupancy to the malic enzyme active site, 60 Å away. We demonstrate that allostery is governed by a large-scale rearrangement that rotates the catalytic subunits 70° between the two states, identifying MaeB as a new model enzyme for the study of ligand-induced conformational change. Our work provides the mechanistic basis for metabolic control of hybrid malic enzymes, and identifies inhibition-insensitive variants that may find utility in synthetic biology.
| S-EPMC7902834 | biostudies-literature
Project description:The amount of pulmonary surfactant within type II cells and in the alveolar space, referred to as surfactant pool sizes, are tightly regulated. The molecular pathways that sense and regulate surfactant pool size within the alveolus have not been identified and constitute a fundamental knowledge gap in the field. Our data show that mice with a germline mutation in the orphan G-protein-coupled receptor, GPR116, have a 30-fold accumulation of surfactant phospholipids that causes respiratory distress in adult animals. This phenotype is associated with increased surfactant secretion and induction of the purinergic receptor P2RY2 in young animals, and lipid-laden macrophages and alveolar destruction in older animals. We further demonstrate that GPR116 mRNA expression is developmentally regulated in the murine lung with peak expression at birth when surfactant pool sizes are maximal. Within the lung, GPR116 protein expression is restricted to the apical plasma membrane of alveolar type I and type II epithelial cells.
2013-04-10 | GSE41417 | GEO
Project description:Cellular senescence both protects multicellular organisms from cancer and contributes to their ageing. The pre-eminent tumour suppressor p53 has an important role in the induction and maintenance of senescence, but how it carries out this function remains poorly understood. In addition, although increasing evidence supports the idea that metabolic changes underlie many cell-fate decisions and p53-mediated tumour suppression, few connections between metabolic enzymes and senescence have been established. Here we describe a new mechanism by which p53 links these functions. We show that p53 represses the expression of the tricarboxylic-acid-cycle-associated malic enzymes ME1 and ME2 in human and mouse cells. Both malic enzymes are important for NADPH production, lipogenesis and glutamine metabolism, but ME2 has a more profound effect. Through the inhibition of malic enzymes, p53 regulates cell metabolism and proliferation. Downregulation of ME1 and ME2 reciprocally activates p53 through distinct MDM2- and AMP-activated protein kinase-mediated mechanisms in a feed-forward manner, bolstering this pathway and enhancing p53 activation. Downregulation of ME1 and ME2 also modulates the outcome of p53 activation, leading to strong induction of senescence, but not apoptosis, whereas enforced expression of either malic enzyme suppresses senescence. Our findings define physiological functions of malic enzymes, demonstrate a positive-feedback mechanism that sustains p53 activation, and reveal a connection between metabolism and senescence mediated by p53.
| S-EPMC3561500 | biostudies-literature