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At least 847 records · Page 47Linked to original sources
PubMed1996

Nucleotide sequence and expression of the gene encoding NADP+- dependent glutamate dehydrogenase (gdhA) from Agaricus bisporus.

The gene encoding NADP+-dependent glutamate dehydrogenase (gdhA) was isolated from an Agaricus bisporus recombinant phage lambda library. The deduced amino acid sequence would specify a 457-amino acid protein that is highly homologous in sequence to those derived from previously isolated and characterized genes coding for microbial NADP+-GDH. The open reading frame is interrupted by six introns. None of the introns is located at either one of the positions of the two introns conserved in the corresponding open reading frames of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. Northern analysis suggests that the A. bisporus gdhA gene is transcriptionally regulated and that, unlike the case in ascomycetes, transcription of this gene is repressed upon the addition of ammonium to the culture.

Agaricus↗
PubMed1996

Export of the periplasmic NADP-containing glucose-fructose oxidoreductase of Zymomonas mobilis.

Glucose-fructose oxidoreductase (GFOR) of the gram-negative bacterium Zymomonas mobilis is a periplasmic enzyme with the tightly bound cofactor NADP. The preprotein carries an unusually long N-terminal signal sequence of 52 amino acid residues. A sorbitol-negative mutant strain (ACM3963) was found to be deficient in GFOR activity and was used for the expression of plasmid-borne copies of the wild-type gfo gene or of alleles encoding alterations in the signal sequence of the pre-GFOR protein. Z. mobilis cells with the wild-type gfo allele translocated pre-GFOR, at least partially, via the Sec pathway since CCCP (carboxylcyanide-m-chlorophenylhydrazone; uncoupler of proton motive force) or sodium azide (inhibitor of SecA) abolished the processing of GFOR. A gfo allele with the hydrophobic region of the signal sequence removed (residues 32-46; Delta32-46) led to a protein that was no longer processed, but showed full enzymatic activity (180 U/mg) and had the cofactor NADP firmly bound. A deletion in the n-region of the signal sequence (residues 2-20; Delta2-20) or exchange of the entire GFOR signal sequence with the signal sequence of gluconolactonase of Z. mobilis led to active and processed GFOR. Strain ACM3963 could not grow in the presence of high sugar concentrations (1 M sucrose) unless sorbitol was added. The presence of the plasmid-borne gfo wild-type allele or of the Delta2-20 deletion led to the restoration of growth on media with 1 M sucrose, whereas the presence of the Delta32-46 deletion led to a growth behavior similar to that of strain ACM3963, with no sorbitol formation from sucrose.

Base Sequence↗
PubMed1998

Plant NADP-dependent isocitrate dehydrogenases are predominantly localized in the cytosol.

The isoenzyme patterns of NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) have been investigated in 15 species of higher plants using dietylaminoethyl ion-exchange chromatography and immunological techniques. The obtained results unambiguously demonstrate that the cytosolic enzyme is the predominant form in leaf extracts of all the surveyed plant species. The chloroplastic isoenzyme, previously reported in pea (Pisum sativum L.) leaves (R.D. Chen et al., 1989, Planta 178; 157-163), is a minor form in ferns and dicotyledonous angiosperms and is undetectable in gymnosperms and monocotyledonous angiosperms. Comparison of immunological relatedness suggests that the proteins of cytosolic isoenzymes have been highly conserved in the course of plant evolution. The data support the previously proposed idea that the cytosol is the major site for alpha-ketoglutarate production to be used for nitrogen assimilation.

Cytosol↗
PubMed1990

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: mechanism of O2-inactivation of the enzyme and its stability in the aerobe.

O2-inactivation of pyruvate:NADP+ oxidoreductase from mitochondria of Euglena gracilis was studied in vitro, and a mechanism which consists of two sequential stages was proposed. Initially, the enzyme is inactivated by the direct action of O2 in a process obeying second-order kinetics. Although the catalytic activity for pyruvate oxidation is lost by this initial inactivation, NADPH oxidation with artificial electron acceptors still occurs. Subsequently, a secondary, O2-independent inactivation occurs, rendering the enzyme completely inactive. Pyruvate stimulates the O2-inactivation while CoA and NADP+ protect the enzyme from O2. The O2-inactivation is accelerated by reduction of the enzyme with pyruvate and CoA. Reactivation of the O2-inactivated enzyme was studied in Ar by incubation with Fe2+ in the presence of some other reducing reagent such as dithiothreitol. The evidence obtained indicates that the partially inactivated enzyme, which retains catalytic activity for NADPH oxidation, can be reactivated, but the completely inactivated enzyme is not. When Euglena cells were exposed to 100% O2 the enzyme in the cells was inactivated by O2, but the rate was quite slow compared with that observed in vitro. The enzyme inactivated by O2 in the cells was almost completely reactivated in vitro by incubation with Fe2+ and other reducing reagents in Ar, suggesting that the secondary, O2-independent inactivation does not occur in situ. When the cells were returned to air, reactivation of the O2-inactivated enzyme in the cells began immediately. The enzyme, kept in isolated, intact mitochondria, was stable in air; however, the enzyme was inactivated by O2 when the mitochondria were incubated with a high concentration of pyruvate.

Animals↗
PubMed1986

Chemical modification and cross-linking as probes of regions on ferredoxin involved in its interaction with ferredoxin: NADP reductase.

Ferredoxin which had been modified with glycine ethylester in the presence of a water-soluble carbodiimide to the extent of one carboxyl-group modified per ferredoxin was subjected to peptide mapping in an attempt to locate the site(s) of modification. The peptide mapping was done by HPLC and analysis of the resulting chromatogram allowed assignment of peaks to various segments in the amino acid sequences of the two isozymes of ferredoxin. The modified ferredoxin appeared to be a mixture of ferredoxin derivatives in which modification had occurred in three areas of the molecule. Although unable to identify the specific residues modified, it has been shown that modification is localized in the regions of residues 26-30, 65-70, and 92-94. The possibility that these regions of ferredoxin may define its binding site for ferredoxin: NADP reductase is discussed. Peptide mapping studies on a covalently linked adduct between ferredoxin and ferredoxin: NADP reductase also support these regions of ferredoxin as being important in the interaction between the two proteins.

Amino Acid Sequence↗
PubMed1975

Crystallization and properties of rat liver malate dehydrogenase (decarboxylating) (NADP).

Rat liver malate dehydrogenase (decarboxylating) (NADP) ((L-malate: NADP) oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) was purified and crystallized from medium containing 30 mM Tris-HCl buffer (pH 7.7), 5 mM MgCl2 and 2 mM 2-mercaptoethanol. The enzyme formed rhomboid crystals free from coenzyme, and appeared homogeneous on isoelectric focusing. The crystalline enzyme had an isoelectric point of pH 6.3. Amino acid analysis showed that it contained more acidic amino acids than basic ones.

Amino Acids↗
PubMed1978

Purification of two forms of the associated 3-dehydroquinate hydro-lyase and shikimate:NADP+ oxidoreductase in Phaseolus mungo seedlings.

Two associated enzymes, 3-dehydroquinate hydro-lyase (EC 4.2.1.10) and shikimate:NADP+ oxidoreductase (EC 1.1.1.25), have been purified from Phaseolus mungo seedlings. These enzymes were purified 6900- and 9700-fold, respectively, but they were not separable. Moreover, two activity bands of the shikimate:NADP+ oxidoreductase were detected after polyacrylamide gel electrophoresis and the two peaks also have 3-dehydroquinate hydro-lyase activity. The two forms of the associated enzymes showed only small differences in molecular weight, Km value, pH optimum and the responses to some inhibitors.

Alcohol Oxidoreductases↗
PubMed1987

Crystallization of an NADP+-dependent malic enzyme from rat liver.

Crystals of a tetrameric NADP+-dependent malic enzyme from rat liver have been grown in the presence of NADP+ using the hanging-drop method of vapour diffusion with ammonium sulphate as the precipitant. Measurement of the crystal density and calculation of the values of Vm for different numbers of polypeptide chains in the unit cell indicate that the asymmetric unit of the crystal contains a complete tetramer, allowing the application of non-crystallographic symmetry to the determination of the molecular structure of this enzyme. This structure would provide only the second example for an enzyme involved in oxidative decarboxylation, the other being 6-phosphogluconate dehydrogenase. In addition, then, to providing an insight into the structure-function relationship in malic enzyme, the successful structure determination would permit valuable comparisons to be made between these two and other enzymes with this catalytic activity.

Animals↗
PubMed1980

Changes in stability of isocitrate dehydrogenase (NADP+) during germination of castor bean seeds.

In crude extract of castor bean endosperm, isocitrate dehydrogenase (NADP+) (EC 1.1.1.42) was stable at 57 degrees C at the beginning of seed germination as well as in maturing and dry seeds. The enzyme gradually became less thermostable as germination proceeded and became unstable after 4 days. Extract from 5-day-old endosperm reduced the thermostability of the thermostable enzyme. The destabilizing factor accumulated in the endosperm as germination progressed and was identified as ricinoleate. Ricinoleate destabilized the purified enzyme which was stabilized by isocitrate and Mg2+, but ricinoleate did not affect the activity of NADP+-isocitrate dehydrogenase itself. Stearate, oleate, palmitate and myristate were similar to ricinoleate in their effect on the thermostability of the enzyme. The thermolabile enzyme in the crude extract of 5-day-old endosperm was readily inactivated by trypsin and in low concentrations of buffer. The thermostable enzyme in the crude extract of 2-day-old endosperm was not affected by these treatments. The thermostable enzyme treated with ricinoleate showed the same instabilities as the thermolabile enzyme. The role of ricinoleate in the germinating castor bean endosperm is discussed.

Ricinus communis↗
PubMed1989

Biochemical studies in marine species--I. NADP(+)-dependent isocitrate dehydrogenase from turbot liver (Scophthalmus maximus L.).

1. An NADP+-dependent isocitrate dehydrogenase was extracted from turbot liver. The enzyme required divalent cations (Mg2+ or Mn2+) for its activity but was inhibited by high salt concentrations. 2. The enzyme had an optimum activity in the pH range between 7.5 and 9.0. The enzymic activity increased with temperature (in the range of 5 to 68 degrees C) with an Ea of 23.5 kJ/mol and a Q10 of 1.34. 3. The apparent Km values for the substrates were 6.5 microM for NADP+, 56 microM for Mg2+, 87 microM for Mn2+ and 4.2 and 73.5 microM for the complexes Mg-isocitrate and Mn-isocitrate, respectively. The physiological significance of these results is discussed. 4. The enzyme was inhibited by citrate and adenine nucleotides. The degree of inhibition depended on the relation between the concentrations and that of magnesium. A possible regulating mechanism is proposed.

Adenine Nucleotides↗
PubMed1998

Factors affecting the oligomeric state of NADP-malic enzyme from maize and wheat tissues: a chemical crosslinking study.

The different aggregational states of maize and wheat NADP-malic enzyme as affected by pH, temperature and various metabolites have been studied by the combined use of intersubunit crosslinking and denaturing polyacrylamide gel electrophoresis. The association/dissociation equilibrium is a pH-dependent process: pH values above 8.0 promote the tetramer formation, while lowering the pH shifts the equilibria towards dimers and monomers. Below pH 6.0, most molecules exist as monomers. In the same way, the temperature governs the equilibria between the different oligomeric states. As the temperature is lowered from 42 to 0 degrees C, a progressive dissociation into dimers and monomers is observed. Excess enthalpies are negative in all cases, but the overall process demands an input of Gibb's free energy. Consequently, the protein dissociation is an entropy-driven process. The presence of Mg2+ or glycerol induces aggregation in both enzymes, while increasing the ionic strength produces the opposite effect. The results suggest that changes in the equilibria between monomer, dimer and tetramer of NADP-malic enzyme could be the molecular basis for an effective regulation of the enzyme activity in vivo.

Cross-Linking Reagents↗
PubMed2001

Integration and expression of Sorghum C(4) phosphoenolpyruvate carboxylase and chloroplastic NADP(+)-malate dehydrogenase separately or together in C(3) potato plants(1).

We have integrated two cDNAs expressing Sorghum photosynthetic phosphoenolpyruvate carboxylase (C(4)-PEPC) and NADP-malate dehydrogenase (cpMDH), two key enzymes involved in the primary carbon fixation pathway of NADP-malic enzyme-type C(4) plants, separately or together into a C(3) plant (potato). Analysis of the transgenic plants showed a 1.5-fold increase in PEPC and cpMDH activities compared to untransformed plants. Immunolocalization confirmed an increase at the protein level of these two enzymes in the transgenic plants and indicated that the Sorghum cpMDH was specifically addressed to the chloroplasts of potato mesophyll cells. However, integration of either or both of the cDNAs into the potato genome did not appear to significantly modify either tuber starch grain content or the rate of photosynthetic O(2) production compared to control untransformed plants. The low level of transgene expression probably explains the lack of influence on carbon metabolism and photosynthetic rates. This general observation suggests that some complex mechanism may regulate the level of production of foreign C(4) metabolism enzymes in C(3) plants.

Journal Article↗
PubMed1994

Thermodynamics of ferredoxin binding to ferredoxin:NADP+ reductase and the role of water at the complex interface.

The association of ferredoxin with ferredoxin:NADP+ reductase (both proteins from spinach chloroplasts) was characterized by isothermal titration calorimetry and fluorescence quenching titration. The formation of the complex is mainly driven by a positive entropy change (delta S = 125 +/- 8 J mol-1 K-1). The calorimetric enthalpy of binding is small between 10 and 37 degrees C and either negative or positive, with an inversion temperature near 25 degrees C. The pH dependence of the association constant [Batie, C. J., & Kamin, H. (1981) J. Biol. Chem. 256, 7756-7763] was shown to correlate with the uptake of a single proton by a group exhibiting a heat of protonation of -26 kJ mol-1. This value agrees with the protonation of an imidazole group. Possible residues to become protonated in the complex are His-19 or His-90 of ferredoxin:NADP+ reductase. The temperature dependence of the free energy of binding, delta G, is weak because of the enthalpy-entropy compensation caused by a heat capacity change, delta Cp, of -680 +/- 44 J mol-1 K-1. The favorable binding entropy and the negative delta Cp indicate a large contribution to binding from hydrophobic effects, which seem to originate from dehydration of the protein-protein interface. Dehydration was demonstrated by osmotic stress experiments in which the association constant was found to increase by 2-4-fold in the presence of 52% (w/w) glycerol. The increase in the association constant with osmotic pressure points to the release of several water molecules from the complex interface.

Calorimetry, Differential Scanning↗
PubMed1998

Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli.

Based on the DNA sequence of its structural genes, clustered in the hnd operon, the NADP-reducing hydrogenase of Desulfovibrio fructosovorans is thought to be a heterotetrameric complex in which HndA and HndC constitute the NADP-reducing unit and HndD constitutes the hydrogenase unit, respectively. The weak representativity of the enzyme among cell proteins has prevented its purification. This paper discusses the purification and characterization of the HndA subunit of this unique tetrameric iron hydrogenase overproduced in Escherichia coli. The purified subunit contains 1.7 mol of non-heme iron and 1.7 mol of acid-labile sulfide/mol. EPR analysis of the reduced form of HndA indicates that it contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with values of gx, gy, and gz equal to 1.915, 1.950, and 2. 000, respectively, and a midpoint redox potential of -395 mV. The UV-visible and EPR spectra of the [2Fe-2S] cluster indicate that HndA belongs to the [2Fe-2S] family typified by the Clostridium pasteurianum [2Fe-2S] ferredoxin. The C-terminal sequence of HndA shows 27% identity with the C-terminal sequence of the 25-kDa subunit of NADH: quinone oxidoreductase from Paracoccus denitrificans, 33% identity with the C-terminal sequence of the 24-kDa subunit from Bos taurus complex I, and 32% identity with the entire sequence of C. pasteurianum [2Fe-2S] ferredoxin. The four cysteine residues involved in HndA cluster binding have been tentatively identified on the basis of sequence identity considerations. Evidence of a HndA organization based on two independent structural domains is discussed.

Amino Acid Sequence↗
PubMed2000

A mathematical model of C(4) photosynthesis: The mechanism of concentrating CO(2) in NADP-malic enzyme type species.

A computer model comprising light reactions in PS II and PS I, electron-proton transport reactions in mesophyll and bundle sheath chloroplasts, all enzymatic reactions and most of the known regulatory functions of NADP-ME type C(4) photosynthesis has been developed as a system of differential budget equations for intermediate compounds. Rate-equations were designed on principles of multisubstrate-multiproduct enzyme kinetics. Some of the 275 constants needed (DeltaG(0)' and K (m) values) were available from literature and others (V (m)) were estimated from reported rates and pool sizes. The model provided good simulations for rates of photosynthesis and pool sizes of intermediates under varying light, CO(2) and O(2). A basic novelty of the model is coupling of NADPH production via NADP-ME with ATP production and regulation of the C(3) cycle in bundle sheath chloroplasts. The functional range of the ATP/NADPH ratio in bundle sheath chloroplasts extends from 1.5 to 2.1, being energetically most efficient around 2. In the presence of such stoichiometry, the CO(2) concentrating function can be explained on the basis of two processes: (a) extra ATP consumption for starch and protein synthesis in bundle sheath leads to a faster NADPH and CO(2) import compared with CO(2) fixation in bundle sheath, and (b) the residual photorespiratory activity consumes RuBP by oxygenation, NADPH and ATP and causes the imported CO(2) to accumulate in bundle sheath cells. As a wider application, the model may be used for predicting results of genetic engineering of plants.

Journal Article↗
PubMed2001

Involvement of matrix NADP turnover in the oxidation of NAD-linked substrates by pea leaf mitochondria.

The involvement of the internal rotenone-insensitive NADPH dehydrogenase on the inner surface of the inner mitochondrial membrane [NDin(NADPH)] in the oxidation of strictly NAD+-linked substrates by pea (Pisum sativum L.) leaf mitochondria was measured. As estimated by the inhibition caused by 5 µM diphenyleneiodonium (DPI) in the presence of rotenone to inhibit complex I, the activity of NDin(NADPH) during glycine oxidation (measured both as O2 uptake and as CO2 release) was 40-50 nmol mg-1 protein min-1. No significant activity of NDin(NADPH) could be detected during the oxidation of 2-oxoglutarate, another strictly NAD+-linked substrate; this was possibly due to its relatively low oxidation rate. Control experiments showed that, even at 125 µM, DPI had no effect on the activity of glycine decarboxylase complex (GDC) and lipoamide dehydrogenase. The relative activity of complex I, NDin(NADPH), and NDin(NADH) during glycine oxidation, estimated using rotenone and DPI, differed depending on the pyridine nucleotide supply in the mitochondrial matrix. This was shown by loading the mitochondria with NAD+ and NADP+, both of which were taken up by the organelle. We conclude that the involvement of NADP turnover during glycine oxidation is not due to the direct production of NADPH by GDC but is an indirect result of this process. It probably occurs via the interconversion of NADH to NADPH by the two non-energy-linked transhydrogenase activities recently identified in plant mitochondria.

Journal Article↗
PubMed2005

Characterization of a novel NADP(+)-dependent D-arabitol dehydrogenase from the plant pathogen Uromyces fabae.

We have identified and characterized a novel NADP(+)-dependent D-arabitol dehydrogenase and the corresponding gene from the rust fungus Uromyces fabae, a biotrophic plant pathogen on broad bean (Vicia faba). The new enzyme was termed ARD1p (D-arabitol dehydrogenase 1). It recognizes D-arabitol and mannitol as substrates in the forward reaction, and D-xylulose, D-ribulose and D-fructose as substrates in the reverse reaction. Co-factor specificity was restricted to NADP(H). Kinetic data for the major substrates and co-factors are presented. A detailed analysis of the organization and expression pattern of the ARD1 gene are also given. Immunocytological data indicate a localization of the gene product predominantly in haustoria, the feeding structures of these fungi. Analyses of metabolite levels during pathogenesis indicate that the D-arabitol concentration rises dramatically as infection progresses, and D-arabitol was shown in an in vitro system to be capable of quenching reactive oxygen species involved in host plant defence reactions. ARD1p may therefore play an important role in carbohydrate metabolism and in establishing and/or maintaining the biotrophic interaction in U. fabae.

Basidiomycota↗
PubMed1985

Purification and some properties of glyoxylate reductase (NADP+) and its functional location in mitochondria in Euglena gracilis z.

Euglena mitochondria contain both glyoxylate reductase (NADP+) and glycollate dehydrogenase to constitute the glycollate-glyoxylate cycle [Yokota & Kitaoka (1979) Biochem. J. 184, 189-192]. Euglena glyoxylate reductase (NADP+) was purified and its submitochondrial location was determined in order to elucidate the cycle. The purified glyoxylate reductase was homogeneous on polyacrylamide-gel electrophoresis. Difference spectra of the purified enzyme revealed that the enzyme was a flavin enzyme. The Mr of the enzyme was 82 000. The enzyme was specific for NADPH, with an apparent Km of 3.9 microM, and for glyoxylate, with an apparent Km of 45 microM. It was 30% as active with oxaloacetate as with glyoxylate. NADH and hydroxypyruvate did not support the activity at all. The optimum pH was 6.45. Submitochondrial fractionation of purified mitochondria showed that the enzyme was located in the intermembrane space and loosely bound to the outer surface of the inner membrane. These properties and the submitochondrial localization of NADPH-glyoxylate reductase facilitate the operation of the glycollate-glyoxylate cycle in combination with glycollate dehydrogenase, which is tightly bound to the inner membrane of Euglena mitochondria.

Alcohol Oxidoreductases↗