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PubMed1995

Effect of adrenaline and phorbol myristate acetate or bacterial lipopolysaccharide on stimulation of pathways of macrophage glucose, glutamine and O2 metabolism. Evidence for cyclic AMP-dependent protein kinase mediated inhibition of glucose-6-phosphate dehydrogenase and activation of NADP+-dependent 'malic' enzyme.

Adrenaline has recently been shown to stimulate both glucose metabolism and H2O2 release by macrophages but the activity of the key pentose phosphate pathway enzyme, glucose-6-phosphate dehydrogenase (which generates the NADPH crucial for the reduction of molecular oxygen), was reduced under these conditions [Costa Rosa, Safi, Cury and Curi (1992) Biochem. Pharmacol. 44, 2235-2241]. We report here that adrenaline activates another NADPH-producing enzyme, NADP(+)-dependent 'malic' enzyme, while also inhibiting glucose-6-phosphate dehydrogenase, via cyclic AMP-dependent protein kinase (PKA) activation. Regulation of glucose-6-phosphate dehydrogenase activity by PKA has not been reported elsewhere. The sparing of some glucose from pentose phosphate pathway consumption may be important in the provision of glycerol 3-phosphate which in the macrophage may be required for new phospholipid synthesis. Glutamine oxidation was also stimulated by adrenaline thus providing increased substrate (malate) for NADP(+)-dependent 'malic' enzyme and therefore shifting some of the burden of NADPH production from glucose to glutamine metabolism. We also report a novel synergistic effect of adrenaline and some bacterial products and/or gamma-interferon in stimulating secretory and metabolic pathways in macrophages which may be a part of a larger network of signals that lead to enhanced macrophage activity.

Animals↗
PubMed2002

Characterization of a Saccharomyces cerevisiae NADP(H)-dependent alcohol dehydrogenase (ADHVII), a member of the cinnamyl alcohol dehydrogenase family.

A new NADP(H)-dependent alcohol dehydrogenase (the YCR105W gene product, ADHVII) has been identified in Saccharomyces cerevisiae. The enzyme has been purified to homogeneity and found to be a homodimer of 40 kDa subunits and a pI of 6.2-6.4. ADHVII shows a broad substrate specificity similar to the recently characterized ADHVI (64% identity), although they show some differences in kinetic properties. ADHVI and ADHVII are the only members of the cinnamyl alcohol dehydrogenase family in yeast. Simultaneous deletion of ADH6 and ADH7 was not lethal for the yeast. Both enzymes could participate in the synthesis of fusel alcohols, ligninolysis and NADP(H) homeostasis.

Alcohol Dehydrogenase↗
PubMed1999

The efficient export of NADP-containing glucose-fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding.

The periplasmic, NADP-containing glucose-fructose oxidoreductase of the gram-negative bacterium Zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twin-arginine motif. In this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose-fructose oxidoreductase to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild-type glucose-fructose oxidoreductase, bind NADP in a looser and dissociable manner, were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of glucose-fructose oxidoreductase into the periplasm uses a Sec-independent apparatus which recognizes, as an additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore, these results suggest that glucose-fructose oxidoreductase is exported in a folded form. A glucose-fructose oxidoreductase:beta-galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of the cytosolic preform of wild-type glucose-fructose oxidoreductase expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the glucose-fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.

Arginine↗
PubMed1973

Inhibition of the oxidation of glutamate and isocitrate in liver mitochondria at a specific NADP+-reducing site.

The cation-complexing carboxylic-acid antibiotic X-537A, at concentrations far below that required for ionophorous activity, selectively inhibits the oxidation of glutamate and isocitrate by liver mitochondria in steady-state 3. The site of inhibition has been localized specifically at the reduction of NADP(+). Glutamate and isocitrate dehydrogenases, the oxidation of NAD(+)-dependent substrates, pyridine nucleotide transhydrogenations, and the respiratory chain between NADH (or NADPH) and O(2) are unaffected by the antibiotic X-537A. Kinetic evidence, i.e., competition between chlorotetracycline (a fluorescence probe for membrane-bound bivalent cation) and X-537A, indicates that the NADP(+)-reducing, antibiotic-sensitive site is most probably associated with the inner mitochondrial membrane.

Adenosine Diphosphate↗
PubMed1974

Amino-acid sequence of NADP-specific glutamate dehydrogenase of neurospora crassa.

A tentative primary structure of the NADP-specific glutamate dehydrogenase [L-glutamate: NADP oxidoreductase (deaminating), EC 1.4.1.4] from Neurospora crassa has been determined. The proposed sequence contains 452 amino-acid residues in each of the identical subunits of the hexameric enzyme. Comparison of the sequence with that of the bovine liver enzyme reveals considerable homology in the amino-terminal portion of the chain, including the vicinity of the reactive lysine, with only shorter stretches of homology within the carboxyl-terminal regions. The significance of this distribution of homologous regions is discussed.

Amino Acid Sequence↗
PubMed1999

Structure of the ternary complex of human 17beta-hydroxysteroid dehydrogenase type 1 with 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin) and NADP+.

Excess 17beta-estradiol (E2), the most potent of human estrogens, is known to act as a stimulus for the growth of breast tumors. Human estrogenic 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), which catalyzes the reduction of inactive estrone (E1) to the active 17beta-estradiol in breast tissues, is a key enzyme responsible for elevated levels of E2 in breast tumor tissues. We present here the structure of the ternary complex of 17beta-HSD1 with the cofactor NADP+ and 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin), an equine estrogen used in estrogen replacement therapy. The ternary complex has been crystallized with a homodimer, the active form of the enzyme, in the asymmetric unit. Structural and kinetic data presented here show that the 17beta-HSD1-catalyzed reduction of E1 to E2 in vitro is specifically inhibited by equilin. The crystal structure determined at 3.0-A resolution reveals that the equilin molecule is bound at the active site in a mode similar to the binding of substrate. The orientation of the 17-keto group with respect to the nicotinamide ring of NADP+ and catalytic residues Tyr-155 and Ser-142 is different from that of E2 in the 17beta-HSD1-E2 complex. The ligand and substrate-entry loop densities are well defined in one subunit. The substrate-entry loop adopts a closed conformation in this subunit. The result demonstrates that binding of equilin at the active site of 17beta-HSD1 is the basis for inhibition of E1-to-E2 reduction by this equine estrogen in vitro. One possible outcome of estrogen replacement therapy in vivo could be reduction of E2 levels in breast tissues and hence the reduced risk of estrogen-dependent breast cancer.

Amino Acid Sequence↗
PubMed1997

An internal cysteine is involved in the thioredoxin-dependent activation of sorghum leaf NADP-malate dehydrogenase.

The chloroplastic NADP-malate dehydrogenase is activated by thiol/disulfide interchange with reduced thioredoxins. Previous experiments showed that four cysteines located in specific N- and carboxyl-terminal extensions were implicated in this process, leading to a model where no internal cysteine was involved in activation. In the present study, the role of the conserved four internal cysteines was investigated. Surprisingly, the mutation of cysteine 207 into alanine yielded a protein with accelerated activation time course, whereas the mutations of the three other internal cysteines into alanines yielded proteins with unchanged activation kinetics. These results suggested that cysteine 207 might be linked in a disulfide bridge with one of the four external cysteines, most probably with one of the two amino-terminal cysteines whose mutation similarly accelerates the activation rate. To investigate this possibility, mutant malate dehydrogenases (MDHs) where a single amino-terminal cysteine was mutated in combination with the mutation of both carboxyl-terminal cysteines were produced and purified. The C29S/C365A/C377A mutant MDH still needed activation by reduced thioredoxin, while the C24S/C365A/C377A mutant MDH exhibited a thioredoxin-insensitive spontaneous activity, leading to the hypothesis that a Cys24-Cys207 disulfide bridge might be formed during the activation process. Indeed, an NADP-MDH where the cysteines 29, 207, 365, and 377 are mutated yielded a permanently active enzyme very similar to the previously created permanently active C24S/C29S/C365A/C377A mutant. A two-step activation model involving a thioredoxin-mediated disulfide isomerization at the amino terminus is proposed.

Catalysis↗
PubMed1999

Structural and enzymatic properties of a gastric NADP(H)- dependent and retinal-active alcohol dehydrogenase.

A class IV-type, gastric alcohol dehydrogenase (ADH) has been purified from frog (Rana perezi) tissues, meaning detection of this enzyme type also in nonmammalian vertebrates. However, the protein is unique among vertebrate ADHs thus far characterized in having preference for NADP(+) rather than NAD(+). Similarly, it deviates structurally from other class IV ADHs and has a phylogenetic tree position outside that of the conventional class IV cluster. The NADP(+) preference is structurally correlated with a replacement of Asp-223 of all other vertebrate ADHs with Gly-223, largely directing the coenzyme specificity. This residue replacement is expected metabolically to correlate with a change of the reaction direction catalyzed, from preferential alcohol oxidation to preferential aldehyde reduction. This is of importance in cellular growth regulation through retinoic acid formed from retinol/retinal precursors because the enzyme is highly efficient in retinal reduction (k(cat)/K(m) = 3.4.10(4) mM(-1) min(-1)). Remaining enzymatic details are also particular but resemble those of the human class I/class IV enzymes. However, overall structural relationships are distant (58-60% residue identity), and residues at substrate binding and coenzyme binding positions are fairly deviant, reflecting the formation of the new activity. The results are concluded to represent early events in the duplicatory origin of the class IV line or of a separate, class IV-type line. In both cases, the novel enzyme illustrates enzymogenesis of classes in the ADH system. The early origin (with tetrapods), the activity (with retinoids), and the specific location of this enzyme (gastric, like the gastric and epithelial location of the human class IV enzyme) suggest important functions of the class IV ADH type in vertebrates.

Alcohol Dehydrogenase↗
PubMed2002

Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo.

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.

Amino Acid Sequence↗
PubMed2004

Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism.

NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP(+)-dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor gamma were markedly elevated. The hepatic and epididymal fat pad contents of acetyl-CoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.

3T3-L1 Cells↗
PubMed2003

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase.

Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.

3T3 Cells↗
PubMed1991

Purification of ferredoxin-NADP+ reductase, flavodoxin and ferredoxin from a single batch of the cyanobacterium Anabaena PCC 7119.

Methods are described for the simultaneous isolation of ferredoxin-NADP+ reductase, ferredoxin and flavodoxin from large quantities of the cyanobacterium Anabaena PCC 7119 allowing the use of a single batch of cells. The ultraviolet-visible spectra and the extinction coefficients of ferredoxin-NADP+ reductase and ferredoxin were determined. The purification procedure also yields enriched fractions of phycobiliproteins and cytochrome c553.

Anabaena↗
PubMed1968

Enzyme activities during the asexual cycle of Neurospora crassa. II. NAD- and NADP-dependent glutamic dehydrogenases and nicotinamide adenine dinucleotidase.

Three enzymes, (a) nicotinamide adenine diphosphate-dependent glutamic dehydrogenase (NAD enzyme), (b) nictoinamide adenine triphosphate-dependent glutamic dehydrogenase (NADP enzyme), and (c) nicotinamide-adenine dinucleotidase (NADase), were measured in separate extracts of Neurospora crassa grown in Vogel's medium N and medium N + glutamate. Specific activities and total units per culture of each enzyme were determined at nine separate intervals phased throughout the asexual cycle. The separate dehydrogenases were lowest in the conidia, increased slowly during germination, and increased rapidly during logarithmic mycelial growth. The amounts of these enzymes present during germination were small when compared with those found later during the production of the conidiophores. The NAD enzyme may be necessary for pregermination synthesis. The NADP-enzyme synthesis was associated with the appearance of the germ tube. Although higher levels of the dehydrogenases in the conidiophores resulted in more enzyme being found in the differentiated conidia, the rate of germination was uneffected. The greatest activity for the NADase enzyme was associated with the conidia, early phases of germination, and later production of new conidia. NADase decreased significantly with the onset of logarithmic growth, remained low during the differentiation of conidiophores, and increased considerably as the conidiophores aged.

Glutamate Dehydrogenase↗
PubMed2001

Structures of F420H2:NADP+ oxidoreductase with and without its substrates bound.

Cofactor F420 is a 5'-deazaflavin derivative first discovered in methanogenic archaea but later found also to be present in some bacteria. As a coenzyme, it is involved in hydride transfer reactions and as a prosthetic group in the DNA photolyase reaction. We report here for the first time on the crystal structure of an F420-dependent oxidoreductase bound with F420. The structure of F420H2:NADP+ oxidoreductase resolved to 1.65 A contains two domains: an N-terminal domain characteristic of a dinucleotide-binding Rossmann fold and a smaller C-terminal domain. The nicotinamide and the deazaflavin part of the two coenzymes are bound in the cleft between the domains such that the Si-faces of both face each other at a distance of 3.1 A, which is optimal for hydride transfer. Comparison of the structures bound with and without substrates reveals that of the two substrates NADP has to bind first, the binding being associated with an induced fit.

Binding Sites↗
PubMed1988

Purification and characterization of NADP+-dependent 3 alpha-hydroxysteroid dehydrogenase from mouse liver cytosol.

A monomeric 3 alpha-hydroxysteroid dehydrogenase with a molecular weight of 34,000 was purified to apparent homogeneity from mouse liver cytosol. The enzyme catalyzed the reversible oxidation of the 3 alpha-hydroxy group of C19-, C21-, and C24-steroids, reduced a variety of carbonyl compounds, and was inhibited by SH-reagents, synthetic estrogens, anti-inflammatory drugs, prostaglandins, and delta 4-3-ketosteroids. Although these properties are similar to those of the enzyme from rat liver cytosol, the mouse enzyme exhibited low dehydrogenase activity toward benzene dihydrodiol and some alicyclic alcohols, it showed a strict cofactor specificity for NADP(H), and high substrate inhibition was observed in the reverse reaction. In addition, dexamethasone, deoxycorticosterone, and medroxyprogesterone acetate inhibited the mouse enzyme competitively at low concentrations and noncompetitively at high concentrations, whereas hexestrol, indomethacin, and prostaglandin A1 were competitive inhibitors. Steady-state kinetic measurements in both directions indicated that the reaction proceeds through an ordered bi bi mechanism with the cofactors binding to the free enzyme. The 3-ketosteroid substrates inhibited the enzyme uncompetitively at elevated concentrations, suggesting that the substrates bind to the enzyme.NADPH complex and to the enzyme NADP+ complex.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Kinetics of hydroperoxide degradation by NADP-glutathione system in mitochondria.

Hydroperoxide decomposition by the NADP-glutathione system in rat liver mitochondria was analyzed. Mitochondria were found to contain high concentrations of the reduced form of glutathione (GSH) (4.32 +/- 0.50 nmol/mg) and NADPH (4.74 +/- 0.64 nmol/mg), and high activities of glutathione peroxidase and reductase. In the initial phase of the reaction, the rate of hydroperoxide decomposition was proportional to both the GSH level and the activity of GSH peroxidase. However, in the later steady state, the step of NADP reduction was rate-limiting, and the overall reaction rate was independent of the initial concentration of GSH, and activities of glutathione peroxidase and reductase. Some GSH was released from mitochondria during incubation, but the rate of the decomposition could be simply expressed as kappa [GSH]/2, where kappa is the first-order rate constant of the peroxidase and [GSH] is the intramitochondrial level of GSH in the steady state. The rate of the reaction in the steady state was also dependent on the NADPH level, its reciprocal being linearly correlated with [NADPH]-1. The rate of decomposition of hydroperoxide was influenced by the respiratory state. During state 3 respiration, the rate was greatly depressed, but was still considered to exceed by far the rate of physiological generation of hydroperoxide.

Animals↗
PubMed1978

Ferredoxin-Sepharose 4B as a tool for the purification of ferredoxin-NADP+ reductase.

Ferredoxin immobilized on Sepharose 4B was prepared by reaction of CNBr-Sepharose 4B with spinach ferredoxin. The ferredoxin-Sepharose 4B conjugated ferredoxin-NADP+ reductase (NADPH: ferredoxin oxidoreductase, [EC 1.6.7.1]) in dilute buffer solution and released it in high salt concentrations. A novel method of preparation for the reductase was established by a combination of affinity adsorption on the ferredoxin-Sepharose 4B column with usual purification procedures. It was found using the new method, that there are two forms of ferredoxin-NADP+ reductase, FNR I and FNR II, in spinach. Comparative studies of the two components suggest that FNR I may be a dimer of FNR II.

Chromatography, Affinity↗
PubMed1978

Studies on the ferredoxin-ferredoxin-NADP reductase complex: kinetic and solvent perturbation studies on the location of sulfhydryl and aromatic amino acid residues.

The molecular weight of spinach ferredoxin-NADP reductase [EC 1.6.99.4] was estimated to be 33,100 by the sedimentation equilibrium method. On the basis of this molecular weight, the amino acid composition of the reductase was determined. The reactivity of ferredoxin toward p-chloromercuribenzoate was investigated. By measuring the time course of the reaction, 1 mol of ferredoxin was found to react with about 8 mol of p-chloromercuribenzoate in 10 min. Under low ionic strength conditions (1 mM NaCl), the second-order rate constants of this reaction determined spectrophotometrically at 420 and 250 nm were 3,640 and 3,690 M-1.S-1, respectively; under high ionic strength conditions (100 mM NaCl), these rate constants were 1,360 and 1,270 M-1.S-1, respectively. In the presence of the reductase, the rate constants under low and high ionic strength conditions were 54 and 1,040 M-1.S-1, respectively. By investigation of the solvent perturbation effects on the aromatic amino acid residues with 20% ethylene glycol, it was found that ferredoxin, ferredoxin-NADP reductase, and the complex between these proteins had 2.8, 6.3, and 3.8 mol of exposed tyrosyl residues per mol of protein, respectively. It therefore seems likely that about 5 tyrosyl residues may exist in the neighborhood of the binding site of the complex of these proteins.

Amino Acids↗