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)

NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been purified to electrophoretic homogeneity from Synechococcus PCC 7942 cells. The native enzyme had a molecular mass of 160 kDa and consisted of four subunits with a molecular mass of 41 kDa. The activity was 6-fold higher with NADPH than with NADH; the apparent Km values for NADPH and NADH were 62 +/- 4.5 and 420 +/- 10.5 microM respectively. The gene encoding NADP-dependent GAPDH was cloned from the chromosomal DNA of Synechococcus 7942. A 1140 bp open reading frame, encoding an enzyme of 380 amino acid residues (approx.molecular mass of 41.3 kDa) was observed. The deduced amino acid sequence of the gene had a greater sequence similarity to the NADP-dependent and chloroplastic form than to the NAD-dependent and cytosolic form. The Synechococcus 7942 enzyme lacked one of the cysteines involved in the light-dependent regulation of the chloroplast enzymes of higher plants. The recombinant enzyme expressed in Escherichia coli as well as the native enzyme purified from Synechococcus 7942 cells were resistant to 1 mM H2O2.
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PMID:Enzymic and molecular characterization of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Synechococcus PCC 7942: resistance of the enzyme to hydrogen peroxide. 868 18

The objective of this study was to determine whether the concentration of pyridine nucleotides in muscle and liver tissue of quail affected the heat stability of aldolase and selected enzymes involved in the oxidation-reduction of these cofactors. The thermal stability of malic enzyme, glyceraldehyde-3-phosphate dehydrogenase, lactic dehydrogenase, and aldolase in liver, and in pectoral muscle of quail was studied at incubation temperatures ranging from 27 to 60 degrees C. The concentrations of liver NAD, NADP, NADPH and the thermal inactivation of liver malic enzyme, glyceraldehyde-3-phosphate dehydrogenase, lactic dehydrogenase, and aldolase were not affected by niacin deficiency. In contrast, pectoral muscle glyceraldehyde-3-phosphate dehydrogenase in the niacin deficient quail compared to that of the controls had a markedly reduced thermal stability. This was associated with a corresponding decrease in the concentration of NAD and possibly NADPH. However, lactic dehydrogenase and aldolase activities were not affected. A similar pattern of heat inactivation was obtained when dialysed muscle and liver extracts were spiked with NAD or NADP. In these studies, NAD(P) protected muscle glyceraldehyde-3-phosphate dehydrogenase against heat inactivation to a much greater degree than that obtained with the other enzymes from muscle or liver tissue. These results suggest a causative relationship between the thermal stability of glyceraldehyde-3-phosphate dehydrogenase and coenzyme status in pectoral muscle tissue. This effect of niacin deficiency on the thermal stability of enzymes appears to be quite selective and specific.
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PMID:Effect of niacin deficiency on the thermal stability of NAD- and NADP-dependent dehydrogenases in liver and pectoral muscle of Japanese quail. 893 Jan 42

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

The function of the membrane-bound ATPase in S. mutans is to regulate cytoplasmic pH values for the purpose of maintaining delta pH. Previous studies have shown that as part of its acid-adaptive ability, S. mutans is able to increase H(+)-ATPase levels in response to acidification. As part of the study of ATPase regulation in S. mutans, we have cloned the ATPase operon and determined its genetic organization. The structural genes from S. mutans were found to be in the order: c, a, b, delta, alpha, gamma, beta, and epsilon; where c and a were reversed from the more typical bacterial organization. The operon contained no I gene homologue but was preceded by a 239-bp intergenic space. Deduced aa sequences from open reading frames indicated that genes encoding homologues of glycogen phosphorylase and nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase flank the H(+)-ATPase operon, 5' and 3' respectively. Sequence analysis indicated the presence of three inverted-repeat nt sequences in the glgP-uncE intergenic space. Primer extension analysis of mRNAs prepared from batch-grown or steady-state cultures demonstrated that the transcriptional start site did not change as a function of culture pH value. The data suggest that potential stem-and-loop structures in the promoter region of the operon do not function to alter the starting position of ATPase-specific mRNA transcription.
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PMID:Cloning and nucleotide sequence analysis of the Streptococcus mutans membrane-bound, proton-translocating ATPase operon. 899 91

Mutations have been introduced in the cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus in order to convert its cofactor selectivity from a specificity towards NAD into a preference for NADP. In the B-S mutant, five mutations (L33T, T34G, D35G, L187A, P188S) were selected on the basis of a sequence alignment with NADP-dependent chloroplastic GAPDHs. In the D32G-S mutant, two of the five mutations mentioned above (L187A, P188S) have been used in combination with another one designed from electrostatic considerations (D32G). Both mutants exhibit a dual-cofactor selectivity at the advantage of either NAD (B-S) or NADP (D32G-S). In order to analyse the cofactor-binding site plasticity at the molecular level, crystal structures of these mutants have been solved, when complexed with either NAD+ (D32G-Sn, resolution 2.5 A, R = 13.9%; B-Sn, 2.45 A, 19.3%) or NADP+ (D32G-Sp, 2.2 A, 19.2%; B-Sp, 2.5 A, 14.4%). The four refined models are very similar to that of the wild-type GAPDH and as expected resemble more closely the holo form than the apo form. In the B-S mutant, the wild-type low affinity for NADP+ seems to be essentially retained because of repulsive electrostatic contacts between the extra 2'-phosphate and the unchanged carboxylate group of residue D32. Such an antideterminant effect is not well compensated by putative attractive interactions which had been expected to arise from the newly-introduced side-chains. In this mutant, recognition of NAD+ is slightly affected with respect to that known on the wild-type, because mutations only weakly destabilize hydrogen bonds and van der Waals contacts originally present in the natural enzyme. Thus, the B-S mutant does not mimic efficiently the chloroplastic GAPDHs, and long-range and/or second-layer effects, not easily predictable from visual inspection of three-dimensional structures, need to be taken into account for designing a true "chloroplastic-like" mutant of cytosolic GAPDH. In the case of the D32G-S mutant, the dissociation constants for NAD+ and NADP+ are practically reversed with respect to those of the wild-type. The strong alteration of the affinity for NAD+ obviously proceeds from the suppression of the two wild-type hydrogen bonds between the adenosine 2'- and 3'-hydroxyl positions and the D32 carboxylate group. As expected, the efficient recognition of NADP+ is partly promoted by the removal of intra-subunit electrostatic repulsion (D32G) and inter-subunit steric hindrance (L187A, P188S). Another interesting feature of the reshaped NADP+-binding site is provided by the local stabilization of the extra 2'-phosphate which forms a hydrogen bond with the side-chain hydroxyl group of the newly-introduced S188. When compared to the presently known natural NADP-binding clefts, this result clearly demonstrates that an absolute need for a salt-bridge involving the 2'-phosphate is not required to switch the cofactor selectivity from NAD to NADP. In fact, as it is the case in this mutant, only a moderately polar hydrogen bond can be sufficient to make the extra 2'-phosphate of NADP+ well recognized by a protein environment.
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PMID:A crystallographic comparison between mutated glyceraldehyde-3-phosphate dehydrogenases from Bacillus stearothermophilus complexed with either NAD+ or NADP+. 917 58

The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. strain PCC 6803, was cloned by functional complementation of an Escherichia coli gap mutant with a genomic DNA library; this is the first time that this cloning strategy has been used for a GAPDH involved in photosynthetic carbon assimilation. The Synechocystis DNA region able to complement the E. coli gap mutant was narrowed down to 3 kb and fully sequenced. A single complete open reading frame of 1,011 bp encoding a protein of 337 amino acids was found and identified as the putative gap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of putative promoter and terminator sites, and orientation of the truncated flanking genes suggested the gap-2 transcript should be monocystronic, a possibility further confirmed by Northern blot studies. Both natural and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic of the native cyanobacterial enzyme, and similar ratios of NAD- to NADP-dependent activities were found in cell extracts from Synechocystis as well as in those from the complemented E. coli clones. The deduced amino acid sequence of Synechocystis GAPDH2 presented a high degree of identity with sequences of the chloroplastic NADP-dependent enzymes. In agreement with this result, immunoblot analysis using monospecific antibodies raised against GAPDH2 showed the presence of the 38-kDa GAPDH subunit not only in crude extracts from the gap-2-expressing E. coli clones and all cyanobacteria that were tested but also in those from eukaryotic microalgae and plants. Western and Northern blot experiments showed that gap-2 is conspicuously expressed, although at different levels, in Synechocystis cells grown in different metabolic regimens, even under chemoheterotrophic conditions. A possible amphibolic role of the cyanobacterial GAPDH2, namely, anabolic for photosynthetic carbon assimilation and catabolic for carbohydrate degradative pathways, is discussed.
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PMID:Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803. 922 60

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

The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyses the irreversible reaction of glyceraldehyde-3-phosphate to 3-phosphoglycerate by the reduction of NADP to NADPH. This is in contrast to the extensively analysed phosphorylating glyceraldehyde-3-phosphate dehydrogenases which catalyse the reversible reaction of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. Sequence analysis revealed that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is not related to the phosphorylating glyceraldehyde-3-phosphate dehydrogenases but a member of the aldehyde dehydrogenase superfamily. The aldehyde dehydrogenases are of ancient origin and they have already existed in the progenote as indicated by phylogenetic analysis. Thus the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase can be found in all three domains, archaea, bacteria and eukarya. The catalytic mechanism of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase and the other aldehyde dehydrogenases resembles a thioester mechanism involving the universally conserved cysteine 298 (pea GAPN). The cofactor of the aldehyde dehydrogenases is bound in a new mode to a structure described as beta-alpha,beta-fold.
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PMID:The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase: biochemistry, structure, occurrence and evolution. 946 40

Cyanobacterial genomes harbour two separate highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, gap1 and gap2, which are closely related at the sequence level to the nuclear genes encoding cytosolic and chloroplast GAPDH of higher plants, respectively. Genes gap1 and gap2 of the unicellular cyanobacterium Synechocystis sp. PCC 6803 were cloned and sequenced and subsequently inactivated by insertional mutagenesis to understand their metabolic functions. We obtained homozygous gap1- mutants which have lost the capacity to grow on glucose under dim light while growth on organic acids as well as photosynthetic growth under CO2 and high light is not impaired. Homozygous gap2- mutants show the reciprocal phenotype. Under dim light they only grow on glucose but not on organic acids nor do they survive under photosynthetic conditions. Measurements of the anabolic activities (reduction of 1,3-bisphosphoglycerate) in extracts from wild type and mutant cells show that Gap2 is a major enzyme with dual cosubstrate specificity for NAD and NADP, while Gap1 displays a minor NAD-specific GAPDH activity. However, if measured in the catabolic direction (oxidation of glyceraldehyde-3-phosphate) Gap2 activity is very low and increases three- to fivefold after gel filtration of extracts over Sephadex G25. Our results suggest that enzymes Gap1 and Gap2, although coexpressed in cyanobacterial wild-type cells, play distinct key roles in catabolic and anabolic carbon flow, respectively. While Gap2 operates in the photosynthetic Calvin cycle and in non-photosynthetic gluconeogenesis, Gap1 seems to be essential only for glycolytic glucose breakdown, conditions under which the catabolic activity of Gap2 seems to be repressed by a specific low-molecular-weight inhibitor.
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PMID:Genetic and biochemical evidence for distinct key functions of two highly divergent GAPDH genes in catabolic and anabolic carbon flow of the cyanobacterium Synechocystis sp. PCC 6803. 948 73

The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction. NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. The coding gene was cloned, sequenced, and expressed in Escherichia coli. Sequence comparisons showed no similarity to phosphorylating glyceraldehyde-3-phosphate dehydrogenases but revealed a relationship to aldehyde dehydrogenases, with the highest similarity to the subgroup of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenases. The activity of the enzyme is affected by a series of metabolites. All effectors tested influence the affinity of the enzyme for its cosubstrate NAD+. Whereas NADP(H), NADH, and ATP reduce the affinity for the cosubstrate, AMP, ADP, glucose 1-phosphate, and fructose 6-phosphate increase the affinity for NAD+. Additionally, most of the effectors investigated induce cooperativity of NAD+ binding. The irreversible catabolic oxidation of glyceraldehyde 3-phosphate, the control of the enzyme by energy charge of the cell, and the regulation by intermediates of glycolysis and glucan degradation identify the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase as an integral constituent of glycolysis in T. tenax. Its regulatory properties substitute for those lacking in the reversible nonregulated pyrophosphate-dependent phosphofructokinase in this variant of the Embden-Meyerhof-Parnas pathway.
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PMID:NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from Thermoproteus tenax. The first identified archaeal member of the aldehyde dehydrogenase superfamily is a glycolytic enzyme with unusual regulatory properties. 949 34


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