NAD and NADP-dependent glutamate dehydrogenase in Hydrogenomonas H 16

Summary Hydrogenomonas H 16 synthetized two chromatographically distinct forms of glutamate dehydrogenase which differed in their thermolability. One glutamate dehydrogenase utilized NAD, the other NADP as a coenzyme.Low specific activity of NAD-dependent glutamate dehydrogenase was found in cells grown with glutamate as sole nitrogen source or in cells grown with a high concentration of ammonium ions. In the presence of a low concentration of ammonium ions or in a nitrogen free medium, the specific activity of the NAD-dependent enzyme increased. Corresponding to the formation of the NAD-dependent glutamate dehydrogenase the enzyme glutamine synthetase was synthesized. The ratio of NAD-dependent glutamate dehydrogenase to glutamine synthetase activity differed only slightly in cells grown with different nitrogen and carbon sources.The NADP-dependent glutamate dehydrogenase was found in high specific activity in cells grown with an excess of ammonium ions. Under nitrogen starvation the formation of the NADP-dependent glutamate dehydrogenase ceased and the enzyme activity decreased.     

Enzymes of nitrogen assimilation in maize roots

The intracellular distribution of enzymes involved in the Crassulacean acid metabolism (CAM) has been studied in Bryophyllum calycinum Salisb. and Crassula lycopodioides Lam. After separation of cell organelles by isopycnic centrifugation, enzymes of the Crassulacean acid metabolism were found in the following cell fractions: Phosphoenolpyruvate carboxylase in the chloroplasts; NAD-dependent malate dehydrogenase in the mitochondria and in the supernatant; NADP-dependent malate dehydrogenase and phosphoenolpyruvate carboxykinase in the chloroplasts; NADP-dependent malic enzyme in the supernatant and to a minor extent in the chloroplasts; NAD-dependent malic enzyme in the supernatant and to some degree in the mitochondria; and pyruvate; orthophosphate dikinase in the chloroplasts. The activity of the NAD-dependent malate dehydrogenase was due to three isoenzymes separated by (NH₄)₂SO₄ gradient solubilization. These isoenzymes represented 17, 78, and 5% of the activity recovered, respectively, in the order of elution. The isoenzyme eluting first was associated with the mitochondria and the second isoenzyme was of cytosolic origin, while the intracellular location of the third isoenzyme was probably the peroxisome. Based on these findings, the metabolic path of Crassulacean acid metabolism within cells of CAM plants is discussed. New address: Institut für Pflanzenphysiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a. D-1000 Berlin 33     

The presence of an NADP-dependent alcohol dehydrogenase activity in Acinetobacter calcoaceticus

Abstract A soluble NADP-dependent alcohol dehydrogenase activity (EC 1.1.1.2) was found in all five strains of Acinetobacter calcoaceticus tested. In A. calcoaceticus NCIB8250, this dehydrogenase was not induced by growth on ethanol, but was present at approximately the same specific activity when this strain was grown on a variety of carbon sources. The specific activity of the NADP-dependent alcohol dehydrogenase is about 10% of the activity of the NAD-dependent alcohol dehydrogenase found in bacteria grown on ethanol. The distinct biochemical properties of the NADP-dependent dehydrogenase showed that this activity was not due to lack of nucleotide specificity of the NAD-dependent dehydrogenase.     

Absence of direct coenzyme transfer in an A-B dehydrogenase system.

Direct transfer of NADPH between two NADP-dependent dehydrogenases, isocitrate dehydrogenase and glutamate dehydrogenase, has been investigated. These enzymes have opposite stereospecificity for hydrogen transfer to the coenzyme. In contrast with the general direct-transfer mechanism postulated for NAD-dependent dehydrogenases [Srivastava & Bernhard (1986) Science 234, 1081-1086], no evidence for direct transfer in either direction was found for these NADP-dependent dehydrogenases.     

NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenases from bacteroides fragilis.

Twenty strains of Bacteroides fragilis were screened for hydroxysteroid oxidoreductase activity in cell-free preparations. Eighteen strains were shown to contain NAD-dependent 7alpha-hydroxysteroid dehydrogenase. Sixteen of the strains containing the NAD-dependent enzyme also contained NADP-depedent 7alpha-hydroxysteroid dehydrogenase, but invariably in lesser amounts. A strain particulary rich in both 7alpha-hydroxysteroid dehydrogenase activities was selected for further study. Measurement of activity as a function of pH revealed a fairly sharp optimal activity range of 9.5--10.0 for the NAD-dependent enzyme and a broad flat optimal range of 7.0--9.0 for the NADP-dependent enzyme. Michaelis constants for trihydroxy-bile acids for the NAD-dependent enzyme were in the range of 0.32--0.34 mM, whereas dihydroxy-bile acids gave a Km of 0.1 mM. Thin-layer chromatography studies on the oxidation product of 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid) by the dehydrogenase revealed a band corresponding to that of synthetic 3alpha-hydroxy, 7-keto-5beta-cholanoic acid. Similarly the oxidation product of chenodeoxycholic acid by both 7alpha-hydroxysteroid dehydrogenase and commercially available 3alpha-hy-droxysteroid dehydrogenase revealed a band corresponding to that of synthetic 3,7-diketo-5beta-cholanoic acid. Neither of these two oxidation products could be distinguished from those by the Escherichia coli dehydrogenase oxidation previously reported. Disc-gel electrophoresis of a cell-free lyophilized preparation indicated one active band for NAD-dependent activity of mobility similar to that for the NADP-dependent E. coli enzyme. The NADP-dependent dehydrogenase was unstable and rapidly lost activity after polyacylamide disc-gel electrophoresis, ultracentrifugation, freezing on refrigeration at 4 degrees C. No 3 alpha- or 12alpha-oriented oxidoreductase activity was demonstrated in any of the strains examined.     

The enzymes of the ammonia assimilation in Pseudomonas aeruginosa

Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen while synthesis of the enzyme was repressed and that present was adenylylated in cultures with excess nitrogen.NADP-and NAD-dependent glutamate dehydrogenase could be separated by column chromatography and showed molecular weights of 110,000 and 220,000, respectively. Synthesis of the NADP-dependent glutamate dehydrogenase is repressed under nitrogen limitation and by growth on glutamate. In contrast, NAD-dependent glutamate dehydrogenase is derepressed by glutamate. Glutamate synthase is repressed by glutamate but not by excess nitrogen.     

An Escherichia coli mutant defective in the NAD-dependent succinate semialdehyde dehydrogenase

Escherichia coli mutants, unable to grown on 4-hydroxyphenylacetate, have been isolated and found to be defective in the NAD-dependent succinate semialdehyde dehydrogenase. When the mutants are grown with 4-aminobutyrate as sole nitrogen source an NAD-dependent succinate semialdehyde dehydrogenase seen in the parental strain is absent but, as in the parental strain, an NADP-dependent enzyme is induced. Growth of the mutants is inhibited by 4-hydroxyphenylacetate due to the accumulation of succinate semialdehyde. The mutants are more sensitive to inhibition by exogenous succinate semialdehyde than is the parental strain. Secondary mutants able to grow in the presence of 4-hydroxyphenylacetate but still unable to use it as sole carbon source were defective in early steps of 4-hydroxyphenylacetate catabolism and so did not form succinate semialdehyde from 4-hydroxyphenylacetate. The gene encoding the NAD-dependent succinate semialdehyde dehydrogenase of Escherichia coli K-12 was located at min 34.1 on the genetic map.     

Alcohol Dehydrogenases from Strawberry Seeds

Two alcohol dehydrogenases (alcohol: NAD oxidoreductase, EC 1.1.1.1 and alcohol: NADP oxidoreductase, EC 1.1.1.2) were partially purified from extracts of strawberry seeds by conventional methods. Some of physical, chemical and kinetic properties of the enzymes are described. On the basis of gel filtration, the molecular weights were estimated to be approximately 78,000 for NAD-dependent enzyme and 82,000 for NADP-dependent enzyme. Thiol-reacting compounds inhibited both enzymes. NAD-dependent alcohol dehydrogenase reacted only with aliphatic alcohols and aldehydes, while aromatic and terpene alcohols and aldehydes were the better substrates for NADP-dependent alcohol dehydrogenase than aliphatic alcohols and aldehydes.     

Enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the female rat.

The segmentation of the proximal tubules in the kidney of the female rat was studied by means of enzyme histochemical reactions and the results compared with those observed in male and recently described by Jacobsen and J0rgensen (1973 a). Reactions were performed for the following soluble, coezyme-dependent oxido-reductases: glucose 6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase, NAD-as well as NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase. Measures were taken to reduce enzyme diffusion and eliminate interference from tissue tetrazolium reductases. Furthermore, reactions were performed for a number of less soluble or insoluble enzymes: glucose 6-phosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases. In the proximal tubules of the female rat all enzymes studied--except beta-hydroxybutyrate dehydrogenase--showed segmental differences, most of them clearly revealing three segments. Sex differences were found concerning all enzymes except uridine diphosphate glucose dehydrogenase and NADP-dependent isocitrate dehydrogenase. The most pronounced sex-related differences were seen in the third segment in which part the male rat showed highest activity in respect to tetrazolium reductases, NAD-dependent isocitrate dehydrogenase, succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase and glucose 6-phosphate dehydrogenase and the female in respect to glucose 6-phosphatase, alpha-glycerophosphate dehydrogenases, and NADP-dependent, decarboxylating malate dehydrogenase. A few of the enzymes exhibited minor sex differences in the first two segments.     

Morphology-associated expression nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase in Mucorracemosus.

The in vivo regulation of glutamate dehydrogenase (GDH) was studied in Mucor racemosus as a function of nutritional conditions and morphological state. Both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP)-dependent GDH activities were found. The effect of carbon and nitrogen source on the specific activity of the NAD-dependent GDH suggests that its role is primarily catabolic. The NAD-dependent activity was generally an order of magnitude greater in mycelial cells than in yeast-phase cells grown on the same medium. During yeast-to-hyphal morphogenesis the increase in NAD-dependent activity preceded the appearance of hyphal cells both under aerobic and anaerobic conditions. Exogenous dibutyryl-cyclic AMP prevented the increase in NAD-dependent GDH concomitantly with the suppression of morphological differentiation. The NADP-dependent activity did not change appreciably during morphogenesis.     

The activities of the NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells are kinetically independent

The bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells has very different kinetic properties from the larger NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase present in all mammalian cells. The NAD-dependent dehydrogenase is unique in that it requires formation of a magnesium.enzyme complex to allow addition of the first substrate, NAD+. It catalyzes an equilibrium ordered kinetic mechanism that has methylenetetrahydrofolate as the last reactant to add and NADH as the last product released. The NADP-dependent dehydrogenase has the same order of addition of substrates, but NADPH is released prior to methenyltetrahydrofolate. The dehydrogenase-cyclohydrolase activities of both enzymes channel methenyltetrahydropteroylglutamate intermediates with the same efficiency which is unaffected by the number of glutamyl residues in the methylenetetrahydrofolate substrate. However, the cyclohydrolase activity of the bifunctional protein is kinetically independent of its dehydrogenase activity, as supported by its lack of inhibition by NAD+, whereas NADP+ strongly inhibits that of the NADP-dependent enzyme. This difference is further demonstrated by the observation that conversion of formyltetrahydrofolate to methylenetetrahydrofolate in the presence of reduced pyridine nucleotide is catalyzed readily only by the bifunctional enzyme.     

¹³C-metabolic enrichment of glutamate in glutamate dehydrogenase mutants of Saccharomyces cerevisiae

Glutamate dehydrogenases (GDH) interconvert α-ketoglutarate and glutamate. In yeast, NADP-dependent enzymes, encoded by GDH1 and GDH3, are reported to synthesize glutamate from α-ketoglutarate, while an NAD-dependent enzyme, encoded by GDH2, catalyzes the reverse. Cells were grown in acetate/raffinose (YNAceRaf) to examine the role(s) of these enzymes during aerobic metabolism. In YNAceRaf the doubling time of wild type, gdh2Δ, and gdh3Δ cells was comparable at ～4 h. NADP-dependent GDH activity (Gdh1p+Gdh3p) in wild type, gdh2Δ, and gdh3Δ was decreased ～80% and NAD-dependent activity (Gdh2p) in wild type and gdh3Δ was increased ～20-fold in YNAceRaf as compared to glucose. Cells carrying the gdh1Δ allele did not divide in YNAceRaf, yet both the NADP-dependent (Gdh3p) and NAD-dependent (Gdh2p) GDH activity was ～3-fold higher than in glucose. Metabolism of [1,2-(13)C]-acetate and analysis of carbon NMR spectra were used to examine glutamate metabolism. Incorporation of (13)C into glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at <15% of wild type. Analysis of (13)C-enrichment of glutamate carbons indicates a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wild type. Further, the relative complexity of (13)C-isotopomers at early time points was noticeably greater in gdh3Δ as compared to wild type and gdh2Δ cells. These in vivo data show that Gdh1p is the primary GDH enzyme and Gdh2p and Gdh3p play evident roles during aerobic glutamate metabolism.     

Genetic control of soluble NDA-dependent sorbitol dehydrogenase in Drosophila melanogaster

Experiments utilizing standard techniques of cell fractionation and disc electrophoresis have revealed the presence of three distinctly different enzymes which catalyze the oxidation of d-sorbitol in crude extracts of Drosophila melanogaster adults. These include (1) a soluble NAD-dependent sorbitol dehydrogenase (NAD-SoDH^s), (2) a mitochondrial NAD-dependent sorbitol dehydrogenase (NAD-SoDH^m), and (3) a soluble NADP-dependent sorbitol dehydrogenase (NADP-SoDH). The structural gene for NAD-SoDH^s has been mapped to a locus between 65.3 and 65.6 on the third chromosome by means of an electrophoretic variant and a low-activity allele. Through the use of segmental aneuploidy, this gene has been localized to the region limited by salivary bands 91B–93F. Because mutants which alter either the activity or electrophoretic mobility of the soluble NAD-dependent enzyme have no significant measurable effect on the mitochondrial or NADP-dependent forms, it is suggested that the enzymes in this system are coded for autonomously by different genes.     

Affinity chromatography of malic enzyme from grape berries

NADP-dependent malic enzyme from grape berries is associated with NAD-dependent malate dehydrogenase. A two step procedure, involving affinity chromatography on 2′,5′-ADP-Sepharose 4B, followed by gel- permeation on Bio-Gel A- 1.5 m, was used to separate malic enzyme from malate dehydrogenase and other proteins. The yield was ca 60% Malic enzyme and malate dehydrogenase migrated respectively as three bands and one band during disc electrophoresis in polyacrylamide gel. The MW resulting from gel-permeation was 220 000 for malic enzyme and 53 000 for malate dehydrogenase.     

Two forms of NADP-dependent malic enzyme in expanding maize leaves

Etiolated maize leaves (Zea mays L.) contain a major isozyme of NADP-dependent malic enzyme (L-malate dehydrogenase, decarboxylating, EC 1.1.1.40) having an isoelectric point of 5.28±0.03, a K_m (L-malate) 0.3–0.6 mM at pH 7.45; a broad pH optimum around pH 6.9 under the conditions of assay; a molecular weight of 280,000 (sometimes accompanied by a minor component of 150,000); and an NAD-dependent activity about 1/50 the NADP-dependent activity. This isozyme, resembling the NADP-malic enzyme of vertebrates, is labeled type 1. The dominant isozyme of young green leaves (type 2) has, however, a pI 4.90±0.03, a K_m (L-malate) 0.10–0.15 mM, a pH optimum of 8, and a molecular weight of 280,000. It is also more stable and exhibits an appreciable NAD-dependent activity (1/5–1/7 the NADP activity). Both isozymes show linear kinetics, dependence on Mn or Mg ions, similar K_m (NADP⁺), and the typical increase of K_m for L-malate with increasing pH values. Type 1 isozyme of maize is assumed to be cytosolic. Type 2 corresponds in each property to the chloroplast enzyme of bundle-sheath cells. It is present at a low level in etiolated leaves and develops to a high specific activity (up to 100 nmol min^-1 mg protein^-1 by 150 h illumination) during photosynthetic differentiation, replacing the type 1 form.Abbreviation MES 2 (N-morpholino)ethane sulfonic acid Work supported by grants from the Consiglio Nazionale delle Ricerche for years 1975 and 1976     

Comparison of isocitrate dehydrogenase from three hyperthermophiles reveals differences in thermostability, cofactor specificity, oligomeric state, and phylogenetic affiliation

With the aim of gaining insight into the molecular and phylogenetic relationships of isocitrate dehydrogenase (IDH) from hyperthermophiles, we carried out a comparative study of putative IDHs identified in the genomes of the eubacterium Thermotoga maritima and the archaea Aeropyrum pernix and Pyrococcus furiosus. An optimum for activity at 90 degrees C or above was found for each IDH. PfIDH and ApIDH were the most thermostable with a melting temperature of 103.7 and 109.9 degrees C, respectively, compared with 98.3 and 98.5 degrees C for TmIDH and AfIDH, respectively. Analytical ultracentrifugation revealed a tetrameric oligomeric state for TmIDH and a homodimeric state for ApIDH and PfIDH. TmIDH and ApIDH were NADP-dependent (K(m)((NADP)) of 55.2 and 44.4 microm, respectively) whereas PfIDH was NAD-dependent (K(m)((NAD)) of 68.3 microm). These data document that TmIDH represents a novel tetrameric NADP-dependent form of IDH and that PfIDH is a homodimeric NAD-dependent IDH not previously found among the archaea. The homodimeric NADP-IDH present in A. pernix is the most common form of IDH known so far. The evolutionary relationships of ApIDH, PfIDH, and TmIDH with all of the available amino acid sequences of di- and multimeric IDHs are described and discussed.     

NAD-dependent malate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase isoenzymes play an important role in dark metabolism of various plastid types

Chloroplasts isolated from spinach (Spinacia oleracea L.) leaves and green sweet-pepper (Capsicum annuum L. var. grossum (L.) Sendt.) fruits contain NADP-dependent malate dehydrogenase (MDH; EC 1.1.1.82) and the bispecific NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13). The NADP-dependent MDH and GAPDH are activated in the light, and inactive in the dark. We found that chloroplasts possess additional NAD-dependent MDH activity which is, like the NAD-dependent GAPDH activity, not influenced by light. In heterotrophic chromoplasts from red sweet-pepper fruits, the NADP-dependent MDH and the NAD(P)-GAPDH isoenzymes disappear during the developmental transition and only NAD-specific isoforms are found. Spinach chloroplasts contain both NAD/H and NADP/H at significant concentrations. Measurements of the pyridine dinucleotide redox states, performed under dark and various light conditions, indicate that NAD(H) is not involved in electron flow in the light. To analyze the contribution of NAD(H)-dependent reactions during dark metabolism, plastids from spinach leaves or green and red sweet-pepper fruits were incubated with dihydroxyacetone phosphate (DHAP). Exogenously added DHAP was oxidized into 3-phosphoglycerate by all types of plastids only in the presence of oxaloacetate, but not with nitrite or in the absence of added electron acceptors. We conclude that the NAD-dependent activity of GAPDH is essential in the dark to produce the ATP required for starch metabolism; excess electrons produced during triose-phosphate oxidation can selectively be used by NAD-MDH to form malate. Thus NADPH produced independently in the oxidative pentose-phosphate pathway will remain available for reductive processes inside the plastids. Received: 2 July 1997 / Accepted: 20 October 1997     

Isolation and characterization of cDNA clones encoding the murine NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase

Forty cDNA clones corresponding to the bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase enzyme were isolated from a mouse lambda gt11 library. Two classes of cDNA clones were shown by Northern analysis to correspond to the two mRNA species of 1.7 and 2.0 kilobases present in transformed cells but not in normal tissues and that apparently are derived from alternate polyadenylation signals. The 1050-base pair coding region encodes a protein of 350 amino acids which contains a putative mitochondrial-targeting signal peptide of 34 amino acids following the initiator methionine. The 20 amino acids immediately following the signal peptide correspond exactly to those determined by sequence analysis of the amino terminus of the purified protein. The derived amino acid sequence of the NAD-dependent dehydrogenase-cyclohydrolase shows extensive homology with the corresponding amino-terminal sequence of the trifunctional NADP-dependent dehydrogenase-cyclohydrolase-synthetase enzyme from human cells (approximately 40%), yeast cytosol (approximately 36%), and yeast mitochondria (approximately 45%).     

Metabolism of l-Malate and d-Malate by a Species of Pseudomonas

Extracts of a fluorescent species of Pseudomonas grown with m-cresol, degrade gentisic acid without isomerization of the ring-fission compound, maleylpyruvate, to give eventually d-malate and pyruvate. d-Malate is also a growth substrate. l-Malate but not d-malate is oxidized by a particulate enzyme not requiring nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP). NAD- or NADP-linked malate dehydrogenases are absent but cells contain an NADP-dependent l-malic enzyme. Exposure of cells to exogenous d-malate induces an NAD-dependent d-malic enzyme, not present when d-malate is formed endogenously. Succinate- or m-cresol-grown cells, containing no d-malic enzyme, rapidly oxidize d-malate in the presence of chloramphenicol at a concentration suffient to inhibit protein synthesis. An NADP-dependent cell-free system, prepared from succinate-grown cells which oxidized d-malate, is described.