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PubMed1983

Influence of Oxygen and Temperature on the Dark Inactivation of Pyruvate, Orthophosphate Dikinase and NADP-Malate Dehydrogenase in Maize.

The influence of oxygen and temperature on the inactivation of pyruvate, Pi dikinase and NADP-malate dehydrogenase was studied in Zea mays. O(2) was required for inactivation of both pyruvate, Pi dikinase and NADP-malate dehydrogenase in the dark in vivo. The rate of inactivation under 2% O(2) was only slightly lower than that at 21% O(2). The in vitro inactivation of pyruvate, Pi dikinase, while dependent on adenine nucleotides (ADP + ATP), did not require O(2).The postillumination inactivation of pyruvate, Pi dikinase in leaves was strongly dependent on temperature. As temperature was decreased in the dark, there was a lag period of increasing length (e.g. at 17 degrees C there was a lag of about 25 minutes) before inactivation proceeded. Following the lag period, the rate of inactivation decreased with decreasing temperature. The half-time for dark inactivation was about 7 minutes at 32 degrees C and 45 minutes at 17 degrees C. The inactivation of pyruvate, Pi dikinase in vitro following extraction from illuminated leaves was also strongly dependent on temperature, but occurred without a lag period. In contrast, NADP-malate dehydrogenase was rapidly inactivated in leaves (half-time of approximately 3 minutes) during the postillumination period without a lag, and there was little effect of temperature between 10 and 32 degrees C. The results are discussed in relation to known differences in the mechanism of activation/inactivation of the two enzymes.

Journal Article↗
PubMed1985

Metabolic Interactions between Spinach Leaf Nitrite Reductase and Ferredoxin-NADP Reductase: Competition for Reduced Ferredoxin.

Steady state rates of NADP reduction decline upon commencement of nitrite reduction in reconstituted chloroplast preparations. Similarly, steady state rates of nitrite reduction are lower, but not zero, during concurrent NADP reduction. These results imply that competition for substrate occurs and suggest that nitrite reduction can successfully compete for reduced ferredoxin, even at high rates of NADP reduction.

Journal Article↗
PubMed1987

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells.

The kinetics of accumulation (per milliliter of culture) of the alpha- and beta- subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The beta-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the alpha-subunit holoenzyme(s). After 120 minutes, the alpha-subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using (35)SO(4) and immunochemical procedures, the rate of synthesis of the alpha-subunit was shown to be greater than the beta-subunit during the first 80 minutes of induction. The alpha- and beta-subunits had different rates of degradation during the induction period (t((1/2)) = 50 versus 150 minutes, respectively) and during the deinduction period (t((1/2)) = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.

Journal Article↗
PubMed1987

Purification and Characterization of Cytosolic NADP Specific Isocitrate Dehydrogenase from Pisum sativum.

Cytosolic NADP-specific isocitrate dehydrogenase was isolated from leaves of Pisum sativum. The purified enzyme was obtained by ammonium sulfate fractionation, ion exchange, affinity, and gel filtration chromatography. The purification procedure yields greater than 50% of the total enzyme activity originally present in the crude extract. The enzyme has a native molecular weight of 90 kilodaltons and is resolved into two catalytically active bands by isoelectric focusing. Purified NADP-isocitrate dehydrogenase exhibited K(m) values of 23 micromolar for dl-isocitrate and 10 micromolar for NADP, and displayed optimum activity at pH 8.5 with both Mg(2+) and Mn(2+).

Journal Article↗
PubMed1990

Purification and Characterization of a Ferredoxin-NADP Oxidoreductase-Like Enzyme from Radish Root Tissues.

An enzyme able to reduce cytochrome c via ferredoxin in the presence of NADPH, was isolated, purified from radish (Raphanus sativus var acanthiformis cultivar miyashige) roots and characterized. The enzyme was purified by DEAE-cellulose, Blue-Cellulofine, Ferredoxin-Sepharose 4B, and Sephadex G-100 column chromatography. Molecular mass of the enzyme was estimated to be 33,000 and 35,000 daltons by Sephadex G-100 gel filtration and SDS-PAGE, respectively. Its absorption spectrum suggested that the enzyme contains flavin as a prosthetic group. The K(m) values for NADPH and ferredoxin were calculated to be 9.2 and 1.2 micromolar, respectively. The enzyme required NADPH and did not use NADH as an electron donor. The optimal pH was 8.4. The enzyme also catalyzed the photoreduction of NADP(+) in the spinach leaf thylakoid membranes depleted of ferredoxin and ferredoxin-NADP(+) oxidoreductase. The effect of NaCl and MgCl(2) concentration on the activity and amino acid composition of the enzyme were demonstrated. The results suggest that the enzyme is similar to ferredoxin-NADP(+) oxidoreductase from chloroplasts and cyanobacteria and is the key enzyme catalyzing the electron transport between NADPH, generated by the pentose phosphate pathway, and ferredoxin in plastids of plant heterotrophic tissues.

Journal Article↗
PubMed2006

Solution structure of HndAc: a thioredoxin-like domain involved in the NADP-reducing hydrogenase complex.

The NADP-reducing hydrogenase complex from Desulfovibrio fructosovorans is a heterotetramer encoded by the hndABCD operon. Sequence analysis indicates that the HndC subunit (52 kDa) corresponds to the NADP-reducing unit, and the HndD subunit (63.5 kDa) is homologous to Clostridium pasteurianum hydrogenase. The role of HndA and HndB subunits (18.8 kDa and 13.8 kDa, respectively) in the complex remains unknown. The HndA subunit belongs to the [2Fe-2S] ferredoxin family typified by C. pasteurianum ferredoxin. HndA is organized into two independent structural domains, and we report in the present work the NMR structure of its C-terminal domain, HndAc. HndAc has a thioredoxin-like fold consisting in four beta-strands and two relatively long helices. The [2Fe-2S] cluster is located near the surface of the protein and bound to four cysteine residues particularly well conserved in this class of proteins. Electron exchange between the HndD N-terminal [2Fe-2S] domain (HndDN) and HndAc has been previously evidenced, and in the present studies we have mapped the binding site of the HndDN domain on HndAc. A structural analysis of HndB indicates that it is a FeS subunit with 41% similarity with HndAc and it contains a possible thioredoxin-like fold. Our data let us propose that HndAc and HndB can form a heterodimeric intermediate in the electron transfer between the hydrogenase (HndD) active site and the NADP reduction site in HndC.

Amino Acid Sequence↗
PubMed1993

Stereospecificity of hydrogen transfer by the NADP-linked alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii.

Class A and class B NAD(H)/NADP(H) coenzyme-dependent dehydrogenases distinguish between the diastereotopic hydrogens pro-R and pro-S at position 4 of the cofactor. We investigated the stereochemistry of hydride transfer in reactions catalyzed by an unusual thermophilic, zinc-containing, NADP-linked enzyme Thermoanaerobium brockii alcohol dehydrogenase (TBAD). Using proton NMR spectroscopy of monodeuterated alcohols and coenzymes we found that TBAD is a class A enzyme that transfers the pro-R hydrogen from the pyridine 4 position of the reduced coenzyme. This stereospecificity is stable over (a) a broad range of temperatures up to 70 degrees C, (b) different concentrations of the coenzyme (catalytic or stoichiometric) and (c) a wide scope of substrates. Although NAD+ is not an effective coenzyme for TBAD, NADP+ and its synthetic analogs, 3-acetylpyridine-ADP+ and thio-NADP+, can be used successfully.

Alcohol Oxidoreductases↗
PubMed1987

Amino acid sequence similarity between malate dehydrogenases (NAD) and pea chloroplast malate dehydrogenase (NADP).

Purified pea chloroplast malate dehydrogenase (NADP) was reduced, S-pyridylethylated with 4-vinyl-pyridine and cleaved with trypsin. The resulting peptides were separated by reversed-phase high-performance liquid chromatography. Several of these peptides were subjected to automated Edman degradation. The sequences obtained were compared to the published primary structures of malate dehydrogenase from the thermophilic bacterium Thermus flavus and with the sequence of heart mitochondrial and cytoplasmic malate dehydrogenase (NAD). Most peptides from choroplast malate dehydrogenase (NADP) showed high homology with sequences of the other malate dehydrogenases, especially with those of the bacterial enzyme. One of the sequenced peptides contains the active-site histidine residue which is conserved in all malate dehydrogenases. Our results suggest a common evolutionary origin for all malate dehydrogenases despite their different coenzyme specificities and regulatory properties. The sequenced peptides which revealed no homology were either located at the amino-terminal or at the carboxy-terminal region of chloroplast malate dehydrogenase (NADP). These novel sequences are most likely plant-specific extensions of an ancestral malate dehydrogenase and may be responsible for the unique light-dependent activation of the chloroplast enzyme.

Amino Acid Sequence↗
PubMed1993

Cloning, site-specific mutagenesis, expression and characterization of full-length chloroplast NADP-malate dehydrogenase from Pisum sativum.

Chloroplast NADP-dependent malate dehydrogenase is regulated by a dithiol redox reaction. The assignment of the groups involved, requires the primary structure of the enzyme to be known. Using the polymerase chain reaction and the cDNA library of Pisum sativum, the sequence of the enzyme and its targeting signal was determined. The gene was cloned in Escherichia coli JM83 and expressed in E. coli JM83 and E. coli B at high yield. The determination of the physical properties of the gene product proves the recombinant protein to be indistinguishable from the enzyme purified from the plant. This holds true, in spite of the fact that the plant enzyme lacks 11 N-terminal residues. The lengths of the complete polypeptide chain of the recombinant enzyme and its transit peptide are 388 and 53 residues, respectively. The comparison of the sequences of the mature enzyme with those of known chloroplast NADP-MDH shows 83-95% identity, but with mitochondrial or bacterial MDH only approximately 20%. Reduction of the (inactive) oxidized enzyme with dithiothreitol allows mimicking of the in vivo activation. The reaction follows a consecutive second-order-kinetics mechanism. Guanidinium chloride (GdmCl) at concentrations below 0.4 M leads to a significant activation of the oxidized form of the enzyme. At [GdmCl] = 0.4-0.46 M, both oxidized and reduced NADP-MDH show highly cooperative changes in the hydrodynamic and spectral properties, indicating the synchronous breakdown of the quaternary, tertiary and secondary structures. Site-directed mutations C23A and C28A do not quench the regulatory properties of the enzyme; additional substitution of alanine for Cys206 and Cys376 renders the enzyme equally active in both the reduced and the oxidized state. Therefore, one can consider these residues, either alone or in combination with Cys23 and Cys28, as responsible for enzyme activation.

Amino Acid Sequence↗
PubMed1995

Kinetics and Thermostability of NADP-Isocitrate Dehydrogenase from Cephalosporium acremonium.

NADP-isocitrate dehydrogenase [isocitrate:NADP(sup+) oxidoreductase (decarboxylating); EC 1.1.1.42] was purified from Cephalosporium acremonium as a single species. The enzyme is a dimer of 140 kDa with identical subunits of 75 kDa. The existence of a monomer-dimer equilibrium is apparent as revealed by an enzyme dilution approach. The chelate complex of the tribasic form of isocitrate and Mg(sup2+) is the true substrate. The V(infmax) depends on a basic form of an ionizable group of the enzyme-substrate complex with a pK(infes) (pK of the enzyme-substrate complex) of 6.9 and a (Delta)H(infion) (activation enthalpy) of -2 (plusmn) 0.4 kcal mol(sup-1) (ca. 8 (plusmn) 2 kJ mol(sup-1)). The enzyme showed maximum activity at 60(deg)C, an unusually high temperature for a nonthermophilic fungus. The thermodynamic parameters for isocitrate oxidative decarboxylation and for the binding of isocitrate and NADP(sup+) were calculated. We analyzed the kinetic thermal stability of the enzyme at pH 6.5 and 7.6. It was inactivated above 40(deg)C following a first-order kinetics. The presence of 12 mM Mg(sup2+) plus 10 mM dl-isocitrate led to 100% protection of enzyme activity against inactivation at 60(deg)C for 120 min. Removal of either or both compounds led to activity loss. A greater stabilizing role for Mg(sup2+) was seen at pH 6.5 than at pH 7.6, whereas the stabilizing effect of isocitrate was not dependent on pH.

Journal Article↗
PubMed1995

Sequence, expression, and function of the gene for the nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase of Streptococcus mutans.

We report the sequencing of a 2,019-bp region of the Streptococcus mutans NG5 genome which contains a 1,428-bp open reading frame (ORF) whose putative translation product had 50% identity to the amino acid sequences of the nonphosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenases (GAPN) from maize and pea. This ORF is located approximately 200 bp downstream of the ptsI gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase transport system. Mutant BCH150, in which the putative gapN gene had been inactivated, lacked GAPN activity that was present in the wild-type strain, thus positively identifying the ORF as the S. mutans gapN gene. Another strain of S. mutans, DC10, which contains an insertionally inactivated ptsI gene, still possessed GAPN activity, as did S. salivarius ATCC 25975, which contains an insertion element between the ptsI and gapN genes. Since the wild-type S. mutans NG5 lacks both glucose-6-phosphate dehydrogenase and NADH:NADP oxidoreductase activities, the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase is important as a means of generating NADPH for biosynthetic reactions.

Amino Acid Sequence↗
PubMed1995

Characterization of an operon encoding an NADP-reducing hydrogenase in Desulfovibrio fructosovorans.

A genomic DNA fragment from Desulfovibrio fructosovorans, which strongly hybridized with the hydAB genes from Desulfovibrio vulgaris Hildenborough, was cloned and sequenced. This fragment was found to contain four genes, named hndA, hndB, hndC, and hndD. Analysis of the sequence homologies indicated that HndA shows 29, 21, and 26% identity with the 24-kDa subunit from Bos taurus complex I, the 25-kDa subunit from Paracoccus denitrificans NADH dehydrogenase type I, and the N-terminal domain of HoxF subunit of the NAD-reducing hydrogenase from Alcaligenes eutrophus, respectively. HndB does not show any significant homology with any known protein. HndC shows 37 and 33% identity with the C-terminal domain of HoxF and the 51-kDa subunit from B. taurus complex I, respectively, and has the requisite structural features to be able to bind one flavin mononucleotide, one NAD, and three [4Fe-4S] clusters. HndD has 40, 42, and 48% identity with hydrogenase I from Clostridium pasteurianum and HydC and HydA from D. vulgaris Hildenborough, respectively. The 4.5-kb length of the transcripts expressed in D. fructosovorans and in Escherichia coli (pSS13) indicated that all four genes were present on the same transcription unit. The sizes of the four polypeptides were measured by performing heterologous expression of hndABCD in E. coli, using the T7 promoter/polymerase system. The products of hndA, hndB, hndC, and hndD were 18.8, 13.8, 52, and 63.4 kDa, respectively. One hndC deletion mutant, called SM3, was constructed by performing marker exchange mutagenesis. Immunoblotting studies carried out on cell extracts from D. fructosovorans wild-type and SM3 strains, using antibodies directed against HndC, indicated that the 52-kDa protein was recognized in extracts from the wild-type strain only. In soluble extracts from D. fructosovorans wild type, a 10-fold induction of NADP reduction was observed when H(2) was present, but no H(2)-dependent NAD reduction ever occurred. This H(2)-dependent NADP reductase activity disappeared completely in extracts from SM3. These results indicate that the hnd operon actually encodes an NAdP-reducing hydrogenase in D. fructosovorans.

Amino Acid Sequence↗
PubMed1985

Cytogenetic localization of a putative regulatory gene affecting an NADP+-dependent enzyme in Drosophila melanogaster.

The cytogenetic localization of a putative regulatory locus affecting an NADP+-dependent enzyme is described. This locus, Mex, is in region 8D10-12 to 9A1-2 on the X chromosome of Drosophila melanogaster. Hyperploidy for this region is associated with significant increases in NADP+-dependent malic enzyme specific activity and specific immunologically cross-reacting material. This is the first report of a specific locus, unlinked to the locus coding for the major polypeptide of the enzyme, which affects an NADP+-dependent enzyme in Drosophila melanogaster.

Animals↗
PubMed1980

Some properties of the NADP-specific malic enzyme from the moderate halophile Vibrio costicola.

NADP-specific malic enzyme (EC 1.1.1.40) has been purified about 160-fold from the moderate halophile Vibrio costicola. The enzyme has a molecular weight of about 120,000. The purified enzyme was unstable in dilute solutions but could be stabilised by NaCl or glycerol. NH4Cl or KCI caused maximal activation at 0.1M, but higher concentrations were inhibitory. NaCl did not activate and was instead a mixed-type inhibitor towards NH4Cl or KCI. The salt concentration affected the kinetic parameters of the reaction. The apparent Km for L-malate reached a minimal value at about 0.1 M salt; the value for NADP, on the other hand, increased continuosly with the Co2+ or Mg2+. NADH was a mixed-type inhibitor towards both substrates, whereas oxaloacetate was strictly competitive towards L-malate and non-competitive towards NADP. The inhibition kinetics were sigmoidal for NADH and hyperbolic for oxaloacetate. The malic enzyme form V. costicola was similar to those of a marine Pseudomonas and Halobacterium cutirubrum in kinetic and regulatory properties but showed a response to salts intermediate between those of the latter enzymes.

Ammonium Chloride↗
PubMed2002

Cloning and expression of Aspergillus niger icdA gene encoding mitochondrial NADP+-specific isocitrate dehydrogenase.

The complementary DNA (cDNA) and chromosomal DNA (icdA) encoding the NADP+-specific isocitrate dehydrogenase (EC 1.1.1.42) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Two cDNA clones (cDNA-1, 2.0 kb; cDNA-2, 1.5 kb) were obtained and sequenced, and an ORF of 1494 base pairs (bp) encoding a protein of 498 amino acids (aa) was identified in cDNA-1. The predicted amino acid sequence showed 73% and 67% sequence identities with those of the mitochondrial NADP+-ICDHs from Saccharomyces cerevisiae and pig, respectively. The sequence analysis of cDNA-1 and -2 revealed that the cDNA-2 lacks a 500-bp fragment from cDNA-1 which contains a mitochondrial targeting motif. A peroxisomal targeting motif at the C-terminus was found on the aa sequences of cDNA-1 and cDNA-2, but the cDNA-2 product seemed to be localized in the cytoplasm since the peroxisomes were not found in the mycelia of WU-2223L cultivated under the conditions of citric acid production. The expression of both cDNAs in Escherichia coli DEK2004, an isocitrate dehydrogenase-deficient mutant, revealed that both cDNAs complemented the glutamate-requiring phenotype, and that the transformants retained NADP(+)-ICDH activities. Therefore, it was clarified that both of the cDNA-1 and -2 products are fully functional. The chromosomal DNA, icdA, was cloned to correspond to cDNA-1, and its nucleotide sequence revealed that it contains seven introns. Southern hybridization using cDNA-1 and cDNA-2 indicated that there is only one copy of icdA on the chromosomes of A. niger WU-2223L. Northern hybridization analysis as for total RNA of WU-2223L revealed that two mRNAs of different sizes, 2.0 kb and 1.5 kb, were hybridized to the ORF of cDNA-1 used as a probe. Therefore, it was found that approximately 1500-nt and 2000-nt mRNAs were transcribed from only one icdA chromosomal gene in A. niger. Such a transcription has not been observed for ICDH, which is one of the key regulatory enzymes in TCA cycle, in any other organisms.

Journal Article↗
PubMedDate not supplied

[Effect of gamma-aminobutyric shunt metabolites on NAD-, NADP-isocitrate dehydrogenases, and aconitate hydratase activities in higher plants].

We studied the influence of metabolites of gamma-aminobutyric shunt of the tricarboxylic acid cycle on the activities of aconitate hydratase (EC.4.2.1.3) as well as NAD- and NADP-specific isocitrate dehydrogenases (EC.1.1.1.41 and EC.1.1.1.42, respectively) using purified enzyme preparations from pea leaves (Pisum sativum L.) and maize scutellum (Zea mays L.). gamma-Aminobutyric acid and succinate proved to have no significant effect on these enzymes, while 0.1-0.2 mM glutamate considerably activated NADP isocitrate dehydrogenase from the both sources, particularly, at unsaturating concentration of the substrate. Succinic semialdehyde stimulated the activities of aconitate hydratase and NADP isocitrate dehydrogenase. The obtained data points to a similar pattern of the effect of intermediates of gamma-aminobutyric shunt on the studied enzymatic activities for both photosynthetic tissues (pea leaves) and those with acidifying, transport, and digestive functions (maize scutellum). However, the absence of pronounced control effects of most metabolites on the studied enzymes allows us to assign them to a relatively inert pool of metabolites.

Aconitate Hydratase↗
PubMed1992

Isolation and characterization of a NADP-dependent glutamate dehydrogenase gene from the primitive eucaryote Giardia lamblia.

Giardia lamblia is believed to be the earliest branching derivative from the eucaryotic lineage. Genomic and cDNA clones encoding the giardia NADP-dependent glutamate dehydrogenase have been isolated and characterized. Southern hydridization using genomic DNA indicates that the gene encoding this activity is unique and single copy. Primer extension, S1 nuclease protection, and genomic and cDNA sequence analysis demonstrate that gene transcripts are initiated within a conserved AT-rich sequence element immediately preceding the ATG translation initiation codon and the short 5' untranslated region is not extended by transsplicing. The open reading frame is 1350 nucleotides in length and encodes a protein of 449 amino acids. The reading frame is not interrupted by introns and the primary transcript is probably not subjected to RNA editing. In the strictly anaerobic metabolism of giardia, NADP-dependent glutamate dehydrogenase activity participates along with alanine aminotransferase, in the cyclic dissipation of reducing equivalents (NADPH) through the conversion of pyruvate to alanine. The deduced amino acid sequence of the giardia protein exhibits substantial homology to numerous fungal and eubacterial NADP-dependent glutamate dehydrogenases. Comparisons of alignment gap positions and amino acid identities indicate that the giardia sequence is at least as similar or more similar to the eubacterial sequence than it is to the fungal sequence. This supports the hypothesis that giardia diverged very early from the eucaryotic lineage.

Amino Acid Sequence↗
PubMed1977

[Comparative study of NADP-reductase properties in two species of purple bacteria].

Unlike Rhodospirillum rubrum, the highly purified preparations of NADP-reductase Thiocapsa roseopersicina are capable of reduction of cytochrome c though they do not catalyse diaphorase reaction in the presence of methyl viologen or benzyl viologen and NADH. T. roseopersicina reductase has more high temperature optimum (50-65 degrees) and more high thermal stability (65 degrees) and it is capable to catalyse diaphorase and menadione-reductase reactions under more high pH values (11.0-12.0) than NADP-reductase of R. rubrum. NADP-reductase of T. roseopersicina is more stable under storing than the enzyme from R. rubrum: the semi-inactivation period of the enzyme when storing in Ar or the air is about 10 and 4 days, respectively, and it takes about three days for R. rubrum.

Argon↗