Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Spinacia oleracea belongs to a wide group of GAPDHs found in most organisms displaying oxygenic photosynthesis, including cyanobacteria, green and red algae, and higher plants. As a major catalytic difference with respect to glycolytic GAPDH, photosynthetic GAPDH exhibits dual cofactor specificity toward pyridine nucleotides with a preference for NADP(H). Here we report the crystal structure of NAD-complexed recombinant A(4)-GAPDH (NAD-A(4)-GAPDH) from Spinacia oleracea, expressed in Escherichia coli. Its superimposition onto native A(4)-GAPDH complexed with NADP (NADP-A(4)-GAPDH) pinpoints specific conformational changes resulting from cofactor replacement. In photosynthetic NAD-A(4)-GAPDH, the side chain of Asp32 is oriented toward the coenzyme to interact with the adenine ribose diol, similar to glycolytic GAPDHs (NAD-specific). On the contrary, in NADP-A(4)-GAPDH Asp32 moves away to accommodate the additional 2'-phosphate group of the coenzyme and to minimize electrostatic repulsion. Asp32 rotation is allowed by the presence of the small residue Ala40, conserved in most photosynthetic GAPDHs, replacing bulky amino acid side chains in glycolytic GAPDHs. While in NADP-A(4)-GAPDH two amino acids, Thr33 and Ser188, are involved in hydrogen bonds with the 2'-phosphate group of NADP, in the NAD-complexed enzyme these interactions are lacking. The crystallographic structure of NAD-A(4)-GAPDH highlights that four residues, Thr33, Ala40, Ser188, and Ala187 (Leu, Leu, Pro, and Leu respectively, in glycolytic Bacillus stearothermophilus GAPDH sequence) are of primary importance for the dual cofactor specificity of photosynthetic GAPDH. These modifications seem to trace the minimum evolutionary route for a primitive NAD-specific GAPDH to be converted into the NADP-preferring enzyme of oxygenic photosynthetic organisms.
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PMID:Dual coenzyme specificity of photosynthetic glyceraldehyde-3-phosphate dehydrogenase interpreted by the crystal structure of A4 isoform complexed with NAD. 1270 26

Specimens of the fruit beetle Pachnoda sinuata were starved for up to 30 days. The weight of the beetles declined consistently throughout the starvation period. Concentrations of carbohydrates and alanine in flight muscles, fat body and haemolymph decreased rapidly after onset of starvation, while the concentration of proline remained high. Whereas the lipid concentrations in the haemolymph did not change significantly upon starvation, the lipid content in flight muscles and fat body decreased significantly.Beetles that had been starved for 14 days responded to injection of Mem-CC, the endogenous neuropeptide from its corpora cardiaca, with hyperprolinaemia and a decrease in the alanine level, but no such effect was monitored after prolonged starvation of 28 days. Regardless of the period of starvation, Mem-CC injection could not cause hypertrehalosaemia or hyperlipaemia, although carbohydrates were increased in fed beetles after injection.Flight ability of beetles that had been starved for 15 or 30 days was apparently not impaired. During such periods, beetles used proline exclusively as fuel for flight as evidenced by the increase in the level of alanine in the haemolymph and decrease of the level of proline; the concentrations of carbohydrates and lipids remained unchanged.Activities of malic enzyme and alanine aminotransferase (enzymes involved in transamination in proline metabolism), glyceraldehyde-3-phosphate dehydrogenase (enzyme of glycolysis), 3-hydroxyacyl-CoA dehydrogenase (enzyme of beta-oxidation of fatty acids) and of malate dehydrogenase (enzyme of Krebs cycle) were measured in fat body and flight muscles. In flight muscle tissue the maximum activity of NAD(+)-dependent malic enzyme increased, while that of glyceraldehyde-3-phosphate dehydrogenase decreased during starvation, and malate dehydrogenase, 3-hydroxyacyl-CoA dehydrogenase and alanine aminotransferase were unchanged. In fat body tissue, activities of NADP(+)-dependent malic enzyme and 3-hydroxyacyl-CoA dehydrogenase increased during food deprivation and activities of glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase and alanine aminotransferase remained unchanged.
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PMID:Metabolic changes in the African fruit beetle, Pachnoda sinuata, during starvation. 1277 Feb 39

Streprococcus pyogenes gapN was cloned and expressed by functional complementation of the Escherichia gap mutant W3CG. The IPTG-induced NADP non-phosphorylating GAPDH (GAPN) has been purified about 75.4 fold from E. coli cells, using a procedure involving conventional ammonium sulfate fractionation, anion-exchange chromatography, hydrophobic chromatography and hydroxyapatite chromatography. The purified protein was characterised: it's an homotetrameric structure with a native molecular mass of 224 kDa, have an acid pI of 4.9 and optimum pH of 8.5. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 60 degrees C with activation energy of 51 KJ mole(-1). The apparent Km values for NADP and D-G3P or DL-G3P were estimated to be 0.385 +/- 0.05 and 0.666 +/- 0.1 mM, respectively and the Vmax of the purified protein was estimated to be 162.5 U mg(-1). The S. pyogenes GAPN was markedly inhibited by sulfydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfydryl groups in the catalytic activity.
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PMID:Purification of recombinant non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus pyogenes expressed in E. coli. 1284 48

NAD-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a cytosolic marker enzyme of eukaryotes (GapC; EC 1.2.1.12). Land plants possess an additional NADP+-dependent enzyme (EC 1.2.1.13) within their chloroplasts which is composed of two subunits, GapA and GapB. Another plastid GAPDH enzyme (GapCp) was recently discovered in gymnosperms and ferns. This novel GapCp is closely related to cytosolic GapC and displays glycolytic NAD+ cosubstrate specificity. Here we show that this new gene GapCp is also present and actively expressed in angiosperms, mosses, and liverworts. Phylogenetic analyses of the available GapC and GapCp sequences suggest that the gene duplication giving rise to GapCp occurred in ancestral charophyte algae. The data are also consistent with a monophyletic origin of charophytes and land plants and further support the view that land plants arose from a Coleochaete-like green alga. Northern hybridizations were employed to study the expression of the genes GapCp, GapC, GapA, and GapB in green and nongreen tissues from pepper (Capsicum annuum). The results demonstrate that GapCp mRNAs are mainly expressed in red pepper fruit and roots, in which the transcript levels of photosynthetic GapA and GapB are downregulated. This suggests that in flowering plants GapCp plays a specific role in glycolytic energy production of nongreen plastids such as chromoplasts and leukoplasts and that angiosperms may be the only land plants where glycolysis is absent in green chloroplasts.
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PMID:Origin, evolution, and metabolic role of a novel glycolytic GAPDH enzyme recruited by land plant plastids. 1296 2

Pentose fermentation to ethanol with recombinant Saccharomyces cerevisiae is slow and has a low yield. A likely reason for this is that the catabolism of the pentoses D-xylose and L-arabinose through the corresponding fungal pathways creates an imbalance of redox cofactors. The process, although redox neutral, requires NADPH and NAD+, which have to be regenerated in separate processes. NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase (ZWF1). To facilitate NADPH regeneration, we expressed the recently discovered gene GDP1, which codes for a fungal NADP+-dependent D-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) (EC 1.2.1.13), in an S. cerevisiae strain with the D-xylose pathway. NADPH regeneration through an NADP-GAPDH is not linked to CO2 production. The resulting strain fermented D-xylose to ethanol with a higher rate and yield than the corresponding strain without GDP1; i.e., the levels of the unwanted side products xylitol and CO2 were lowered. The oxidative part of the pentose phosphate pathway is the main natural path for NADPH regeneration. However, use of this pathway causes wasteful CO2 production and creates a redox imbalance on the path of anaerobic pentose fermentation to ethanol because it does not regenerate NAD+. The deletion of the gene ZWF1 (which codes for glucose-6-phosphate dehydrogenase), in combination with overexpression of GDP1 further stimulated D-xylose fermentation with respect to rate and yield. Through genetic engineering of the redox reactions, the yeast strain was converted from a strain that produced mainly xylitol and CO2 from D-xylose to a strain that produced mainly ethanol under anaerobic conditions.
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PMID:Engineering redox cofactor regeneration for improved pentose fermentation in Saccharomyces cerevisiae. 1453 41

A group of unicellular eukaryotic algae, the dinoflagellates, are known to possess two types of gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An enzyme encoded by one type of gene possibly plays a key role in the glycolytic pathway of the cytosol and the other in the Calvin cycle of plastids. In the present study, an additional type of GAPDH gene (GapC3) was found in the symbiotic dinoflagellates, Symbiodinium spp. and their related species, Gymnodinium simplex and Polarella glacialis, all of which belong to the order Suessiales. Since no intracellular translocation signal is found at both amino- and carboxy-termini of its deduced amino acid sequence, the protein is predicted to function in the cytosol. However, it may not be involved in glycolysis due to the presence of an amino acid signature that allows binding for NADP+. It is likely that dinoflagellate species, other than Suessiales investigated in this study, lack this type of GAPDH. Phylogenetic analysis placed GapC3 from the Suessialean species firmly in the clade composed of GAPDH from spirochetes, euglenophytes (cytosolic type) and kinetoplastids (glycosomal type). Specifically, this enigmatic GAPDH gene in dinoflagellates was closely related to its cytosolic counterpart in euglenophytes. It has been previously reported that plastid-targeted (Calvin cycle) GAPDH genes of the dinoflagellates Pyrocystis spp. and that of the euglenophyte Euglena gracilis also seem to share a common ancestor. It appears highly likely that at least two genes (cytosolic and plastid-targeted GAPDH genes) have been laterally transferred between these two eukaryotic algal groups.
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PMID:An enigmatic GAPDH gene in the symbiotic dinoflagellate genus Symbiodinium and its related species (the order Suessiales): possible lateral gene transfer between two eukaryotic algae, dinoflagellate and euglenophyte. 1465

Immunolocalization experiments indicate that both the subunit B of the NADP-linked chloroplastic glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) and the NAD-linked cytosolic enzyme (EC 1.2.1.12) are present in the pea ( Pisum sativum L.) leaf nucleus. Subunit A of the NADP-linked enzyme appears to be restricted to the chloroplast.
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PMID:Cytosolic glyceraldehyde-3-P dehydrogenase and the B subunit of the chloroplast enzyme are present in the pea leaf nucleus. 1500 41

In green parts of the plant, during illumination ATP and NAD(P)H act as energy sources that are generated mainly in photosynthesis and respiration, whereas in darkness, glycolysis, respiration and the oxidative pentose-phosphate pathway (OPP) generate the required energy forms. In non-green parts, sugar oxidation in glycolysis, respiration and OPP are the only means of producing energy. For energy-consuming reactions, the delivery of NADPH, NADH, reduced ferredoxin and ATP has to take place at the required rates and in the specific compartments, since the pool sizes of these energy carriers are rather limited and, in general, they are not directly transported across biomembranes. Indirect transport of reducing equivalents can be achieved by malateoxaloacetate shuttles, involving malate dehydrogenase (MDH) for the interconversion. Isoenzymes of MDH are present in each cellular compartment. Chloroplasts contain the redox-controlled NADP-MDH that is only active in the light. In addition, a plastid NAD-MDH that is permanently active and is present in all plastid types has been found. Export of excess NAD(P)H through the malate valves will allow for the continued production of ATP (1) in photosynthesis, and (2) in oxidative phosphorylation. In the latter case, the coupled production of NADH is catalysed by the bispecific NAD(P)-GAPDH (GapAB) in chloroplasts that is active with NAD even in darkness, or by the specific plastid NAD-GAPDH (GapCp) in non-green tissues. When plants are subjected to conditions such as high light, high CO(2), NH(4) (+) nutrition, cold stress, which require changed activities of the enzymes of the malate valves, changed expression levels of the MDH isoforms can be observed. In nodules, the induction of a nodule-specific plastid NAD-MDH indicates the changed requirements for energy supply during N(2) fixation. Furthermore, the induction of glucose 6-phosphate dehydrogenase isoforms by ammonium and of ferredoxin and ferredoxin-NADP reductase by nitrate has been described. All these findings are in line with the assumption that a changed redox state caused by metabolic variability leads to the induction of enzymes involved in redox poise.
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PMID:Malate valves to balance cellular energy supply. 1503 73

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.
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PMID:Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin. 1523 65

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of the hyperthermophilic Archaeum Thermoproteus tenax is a member of the superfamily of aldehyde dehydrogenases (ALDH). GAPN catalyses the irreversible oxidation of glyceraldehyde 3-phosphate (GAP) to 3-phosphoglycerate in the modified glycolytic pathway of this organism. In contrast to other members of the ALDH superfamily, GAPN from T.tenax (Tt-GAPN) is regulated by a number of intermediates and metabolites. In the NAD-dependent oxidation of GAP, glucose 1-phosphate, fructose 6-phosphate, AMP and ADP increase the affinity for the cosubstrate, whereas ATP, NADP, NADPH and NADH decrease it leaving, however, the catalytic rate virtually unaltered. As we show here, the enzyme also uses NADP as a cosubstrate, displaying, however, unusual discontinuous saturation kinetics indicating different cosubstrate affinities and/or reactivities of the four active sites of the protein tetramer caused by cooperative effects. Furthermore, in the NADP-dependent reaction the presence of activators decreases the overall S0.5 and increases Vmax by a factor of 3. To explore the structural basis for the different effects of both pyridine nucleotides we solved the crystal structure of Tt-GAPN in complex with NAD at 2.2 A resolution and compared it to the binary Tt-GAPN-NADPH structure. Although both pyridine nucleotides show a similar binding mode, NADPH appears to be more tightly bound to the protein via the 2' phosphate moiety. Moreover, we present four co-crystal structures with the activating molecules glucose 1-phosphate, fructose 6-phosphate, AMP and ADP determined at resolutions ranging from 2.3 A to 2.6 A. These crystal structures reveal a common regulatory site able to accommodate the different activators. A phosphate-binding pocket serves as an anchor point ensuring similar binding geometry. The observed conformational changes upon activator binding are discussed in terms of allosteric regulation. Furthermore, we present a crystal structure of Tt-GAPN in complex with the substrate D-GAP at 2.3 A resolution, which allows us to analyse the structural basis for substrate binding, the mechanism of catalysis as well as the stereoselectivity of the enzymatic reaction.
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PMID:Structural Basis of allosteric regulation and substrate specificity of the non-phosphorylating glyceraldehyde 3-Phosphate dehydrogenase from Thermoproteus tenax. 1528 89


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