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Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
 6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Higher-plant chloroplast NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) is composed of two different nuclear-encoded subunits, GAPA and GAPB, forming the highly active heterotetrameric A2B2 enzyme. The main difference between these two subunits is a C-terminal extension of about 30 amino acid residues of GAPB. We present cDNA clones for a nuclear-encoded chloroplast protein from pea, spinach and tobacco, which we have named CP12. The mature protein consists of only 74, 75 and 76 amino acid residues, respectively and contains two domains with significant homology to the C-terminal extension of GAPB. Affinity chromatography approaches reveal also a specific interaction between CP12 and chloroplast GAPDH. Northern blot analysis indicates that CP12 is, like plastid GAPDH, expressed in green and also in etiolated leaves. Further homology is observed between CP12 and ORF3, an open reading frame located in the hox gene cluster of Anabaena variabilis. This gene cluster encodes the subunits of the bidirectional NADP(+)-dependent [NiFeS] dehydrogenase. We propose therefore a common evolutionary origin of CP12 and higher-plant chloroplast GAPDH subunit GAPB from the cyanobacterial ORF3.
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PMID:CP12: a small nuclear-encoded chloroplast protein provides novel insights into higher-plant GAPDH evolution. 898 May 47

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1. 13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).
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PMID:CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants. 929 36

For higher plant chloroplasts, two key enzymes of the Calvin cycle, phosphoribulokinase (EC 2.7.1.19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13), have recently been shown to be oligomerized onto the nonenzymatic peptide CP12. Enzymatic activity depends on complex dissociation, mediated by NADPH. The discovery of genes for CP12 in mosses, green algae, and cyanobacteria, together with the analysis of equivalent multiprotein complexes of Chlamydomonas and Synechocystis suggests that light regulation of Calvin cycle activity via NADPH-mediated reversible phosphoribulokinase/CP12/GAPDH complex dissociation is conserved in all photosynthetic organisms, prokaryotes and eukaryotes. In vitro complex reconstitution assays with heterologously expressed Synechocystis wild-type and mutagenized CP12 demonstrate a conserved subunit composition, stoichiometry, and topology in this complex. Further finding of genes, coding for chimeric proteins, carrying CP12 or parts of it as genetic fusions, indicates that evolution has used the peptide loops of CP12 as universal modules to keep various enzymatic activities under the control of NADP(H). These fusion events occurred at least twice in evolution. First was the fusion of the duplicated genes for CP12 and the ORF4 protein of Anabaena variabilis to the chimeric gene for the heterocyst-specific expressed ORF3 protein, most probably involved in N2 fixation. A second gene fusion, which led to the higher plant chloroplast-specific GAPDH subunit, GAPB, has taken place during the transition from water- to land plants.
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PMID:Evolutionary conserved light regulation of Calvin cycle activity by NADPH-mediated reversible phosphoribulokinase/CP12/ glyceraldehyde-3-phosphate dehydrogenase complex dissociation. 968 44

Light/dark modulation of the higher plant Calvin-cycle enzymes phosphoribulokinase (PRK) and NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP- GAPDH-A2B2) involves changes of their aggregation state in addition to redox changes of regulatory cysteines. Here we demonstrate that plants possess two different complexes containing the inactive forms (a) of NADP-GAPDH and PRK and (b) of only NADP-GAPDH, respectively, in darkened chloroplasts. While the 550-kDa PRK/GAPDH/CP12 complex is dissociated and activated upon reduction alone, activation and dissociation of the 600-kDa A8B8 complex of NADP-GAPDH requires incubation with dithiothreitol and the effector 1,3-bisphosphoglycerate. In the light, PRK is therefore completely in its activated state under all conditions, even in low light, while GAPDH activation in the light is characterized by a two-step mechanism with 60-70% activation under most conditions in the light, and the activation of the remaining 30-40% occurring only when 1,3-bisphosphoglycerate levels are strongly increasing. In vitro studies with the purified components and coprecipitation experiments from fresh stroma using polyclonal antisera confirm the existence of these two aggregates. Isolated oxidized PRK alone does not reaggregate after it has been purified in its reduced form; only in the presence of both CP12 and purified NADP-GAPDH, some of the PRK reaggregates. Recombinant GapA/GapB constructs form the A8B8 complex immediately upon expression in E. coli.
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PMID:Co-existence of two regulatory NADP-glyceraldehyde 3-P dehydrogenase complexes in higher plant chloroplasts. 1242 61

The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) embedded in the phosphoribulokinase (PRK).GAPDH.CP12 complex was increased 2-3-fold by reducing agents. This occurred by interaction with PRK as the cysteinyl sulfhydryls (4 SH/subunit) of GAPDH within the complex were unchanged whatever the redox state of the complex. But isolated GAPDH was not activated. Alkylation plus mass spectrometry also showed that PRK had one disulfide bridge and three SH groups per monomer in the active oxidized complex. Reduction disrupted this disulfide bridge to give 2 more SH groups and a much more active enzyme. We assessed the kinetics and dynamics of the interactions between PRK and GAPDH/CP12 using biosensors to measure complex formation in real time. The apparent equilibrium binding constant for GAPDH/CP12 and PRK was 14 +/- 1.6 nm for oxidized PRK and 62 +/- 10 nm for reduced PRK. These interactions were neither pH- nor temperature-dependent. Thus, the dynamics of PRK.GAPDH.CP12 complex formation and GAPDH activity are modulated by the redox state of PRK.
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PMID:Modulation, via protein-protein interactions, of glyceraldehyde-3-phosphate dehydrogenase activity through redox phosphoribulokinase regulation. 1255 49

Protein-protein interactions are involved in many metabolic pathways. This review will focus on the role of such associations in CO2 assimilation (Benson-Calvin cycle) and especially on the involvement of a GAPDH/CP12/PRK complex which has been identified in many photosynthetic organisms and may have an important role in the regulation of CO2 assimilation. The emergence of new kinetic and regulatory properties as a consequence of protein-protein interactions will be addressed as well as some of the questions raised by the existence of these supramolecular complexes such as composition, function, and assembly pathways. The presence and role of small intrinsically unstructured proteins like the 8.5 kDa protein CP12, involved in the regulation and/or assembly of these complexes will be discussed.
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PMID:Emergence of new regulatory mechanisms in the Benson-Calvin pathway via protein-protein interactions: a glyceraldehyde-3-phosphate dehydrogenase/CP12/phosphoribulokinase complex. 1504 59

Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) interact in the chloroplast stroma through the action of the small peptide CP12. This supramolecular complex concurs with the light-dependent modulation in vivo of GAPDH and PRK activities. The expression patterns of several genes potentially involved in the formation of the complex have been studied. The genome of Arabidopsis thaliana includes seven genes for phosphorylating GAPDH isozymes, one PRK gene, and three genes for CP12. The expression of four GAPDH genes was analysed, i.e. GapA-1 and GapB for photosynthetic GAPDH of chloroplasts (NAD(P)-dependent), GapC-1 for cytosolic GAPDH, and GapCp-1 for plastid GAPDH (both NAD-dependent). A similar analysis was performed with PRK and two CP12 genes (CP12-1, CP12-2). The expression of GapA-1, GapB, PRK, and CP12-2 was found to be co-ordinately regulated with the same organ specificity, all four genes being mostly expressed in leaves and flower stalks, less expressed in flowers, and little or not expressed in roots and siliques. The expression of all these genes in leaves was terminated during prolonged darkness or following sucrose treatments, and their transcripts decayed with similar kinetics. At variance with CP12-2, gene CP12-1 appeared to be expressed more in flowers, it was totally insensitive to darkness, and less affected by sucrose. The expression of glycolytic GapC was strong and ubiquitous, insensitive to dark treatments, and unaffected by sucrose. GapCp transcripts were also found to be ubiquitous at lower levels, slowly decreasing in the dark and stable in sucrose-treated leaves. The co-ordinated expression of genes GapA-1, GapB, PRK, and CP12-2 is consistent with their specific involvement in the formation of the photosynthetic regulatory complex of chloroplasts.
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PMID:Co-ordinated gene expression of photosynthetic glyceraldehyde-3-phosphate dehydrogenase, phosphoribulokinase, and CP12 in Arabidopsis thaliana. 1553 78

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the chloroplast of Chlamydomonas reinhardtii is part of a complex that also includes phosphoribulokinase (PRK) and CP12. We identified two residues of GAPDH involved in protein-protein interactions in this complex, by changing residues K128 and R197 into A or E. K128A/E mutants had a Km for NADH that was twice that of the wild type and a lower catalytic constant, whatever the cofactor. The kinetics of the mutant R197A were similar to those of the wild type, while the R197E mutant had a lower catalytic constant with NADPH. Only small structural changes near the mutation may have caused these differences, since circular dichroism and fluorescence spectra were similar to those of wild-type GAPDH. Molecular modelling of the mutants led to the same conclusion. All mutants, except R197E, reconstituted the GAPDH-CP12 subcomplex. Although the dissociation constants measured by surface plasmon resonance were 10-70-fold higher with the mutants than with wild-type GAPDH and CP12, they remained low. For the R197E mutation, we calculated a 4 kcal/mol destabilizing effect, which may correspond to the loss of the stabilizing effect of a salt bridge for the interaction between GAPDH and CP12. All the mutant GAPDH-CP12 subcomplexes failed to interact with PRK and to form the native complex. The absence of kinetic changes of all the mutant GAPDH-CP12 subcomplexes, compared to wild-type GAPDH-CP12, suggests that mutants do not undergo the conformation change essential for PRK binding.
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PMID:Involvement of two positively charged residues of Chlamydomonas reinhardtii glyceraldehyde-3-phosphate dehydrogenase in the assembly process of a bi-enzyme complex involved in CO2 assimilation. 1560 60

In Synechococcus PCC7942 cells grown in the dark, the concentrations of NAD(H) and NADP(H) were 128+/-2.5 and 483+/-4.0 microm, respectively, while those in the cells under light conditions were 100+/-5.0 and 649+/-7.0 microm, respectively. Analysis of gel filtration indicated that the change of the ratio of NADP(H) to NAD(H) in cyanobacterial cells under light/dark conditions controls the reversible dissociation of the PRK/CP12/GAPDH complex (approximately 520 kDa) consisting of phosphoribulokinase (PRK), CP12, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). S. 7942 CP12 lacked the two Cys residues essential for formation of the N-terminal peptide loop in the CP12 of higher plants, but the N-terminal region of S. 7942 CP12 had the ability to be associated with PRK. The growth of mutant cells in which the CP12 gene was disrupted by a kanamycin resistance cartridge gene was almost the same as that of wild-type cells under continuous light conditions. However, under the light/dark cycle (12 h/12 h), the growth of CP12-disrupted mutant cells was significantly inhibited compared with that of wild-type cells. The mutant cells showed a decreased rate of O2 consumption and an increased level of ribulose 1,5-bisphosphate compared with wild-type cells in the dark. These data suggest that under light and dark conditions, the oligomerization of CP12 with PRK and GAPDH regulates the activities of both enzymes and thus the carbon flow from the Calvin cycle to the oxidative pentose phosphate cycle.
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PMID:The Calvin cycle in cyanobacteria is regulated by CP12 via the NAD(H)/NADP(H) ratio under light/dark conditions. 1586 9

Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form together with the regulatory peptide CP12 a supramolecular complex in Arabidopsis (Arabidopsis thaliana) that could be reconstituted in vitro using purified recombinant proteins. Both enzyme activities were strongly influenced by complex formation, providing an effective means for regulation of the Calvin cycle in vivo. PRK and CP12, but not GapA (A(4) isoform of GAPDH), are redox-sensitive proteins. PRK was reversibly inhibited by oxidation. CP12 has no enzymatic activity, but it changed conformation depending on redox conditions. GapA, a bispecific NAD(P)-dependent dehydrogenase, specifically formed a binary complex with oxidized CP12 when bound to NAD. PRK did not interact with either GapA or CP12 singly, but oxidized PRK could form with GapA/CP12 a stable ternary complex of about 640 kD (GapA/CP12/PRK). Exchanging NADP for NAD, reducing CP12, or reducing PRK were all conditions that prevented formation of the complex. Although GapA activity was little affected by CP12 alone, the NADPH-dependent activity of GapA embedded in the GapA/CP12/PRK complex was 80% inhibited in respect to the free enzyme. The NADH activity was unaffected. Upon binding to GapA/CP12, the activity of oxidized PRK dropped from 25% down to 2% the activity of the free reduced enzyme. The supramolecular complex was dissociated by reduced thioredoxins, NADP, 1,3-bisphosphoglycerate (BPGA), or ATP. The activity of GapA was only partially recovered after complex dissociation by thioredoxins, NADP, or ATP, and full GapA activation required BPGA. NADP, ATP, or BPGA partially activated PRK, but full recovery of PRK activity required thioredoxins. The reversible formation of the GapA/CP12/PRK supramolecular complex provides novel possibilities to finely regulate GapA ("non-regulatory" GAPDH isozyme) and PRK (thioredoxin sensitive) in a coordinated manner.
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PMID:Reconstitution and properties of the recombinant glyceraldehyde-3-phosphate dehydrogenase/CP12/phosphoribulokinase supramolecular complex of Arabidopsis. 1625 9


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