Changes in glutamate dehydrogenase activity of Chlamydomonas reinhardtii under different trophic and stress conditions

Abstract. Under stress conditions (darkness, nitrogen starvation, high ammonium concentrations, glutamine synthetase and glutamate synthase inhibition) glutamate dehydrogenase animating activity levels of Chlamydomonas cells varied inversely to those of glutamine synthetase. Nitrogen and carbon sources also influenced glutamate dehydrogenase levels in Chlamydomonas , the highest values being found in cells cultured mixotrophically with ammonium, under which conditions glutamate dehydrogenase and glutamine synthetase levels were likewise inversely related. These facts, together with the analysis of internal fluctuations of ammonium, 2-oxoglutarate, and the amino acid pool as well as the variations of certain enzymes involved in carbon metabolism indicate that glutamate dehydrogenase animating activity is adaptative, being involved in the maintenance of intracellular levels of L-glutamate when they cannot be maintained by the GS-GOGAT cycle, and probably more connected with carbon than nitrogen metabolism.     

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.     

Ammonium-assimilating enzymes and their regulation in wild and NADP-glutamate dehydrogenase-deficient strains of the ectomycorrhizal fungus Hebeloma cylindrosporum

Hebeloma cylindrosporum strain h 17 was grown on media containing either glutamate or ammonium as nitrogen source. Growth tests and in vitro activity measurements revealed that both glutamine synthetase (GS. EC 6.3.1.2) and NADP-specific glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) are fully functional in wild type mycelia grown on glutamate or ammonium as sole nitrogen source. However, NADP-GDH appeared to be more active than GS in stationary growing mycelia. NADP-GDH is also able to sustain adequate ammonium assimilation in methionine sulfoximine (MSX)-treated mycelia since they grew as well as mycelia fed with ammonium alone. The NADP-GDH also appeared to be L-glutamate inducible whereas GS was repressed by ammonium. The NADP-GDH deficient strain, when transferred from a glutamate containing medium to an ammonium containing medium, exhibited a derepressed GS, although this enzyme did not fully substitute for the deficiency of NADP-GDH in ammonium assimilation. The low NADP-GDH activity of the mutant strain exhibited a reduced mobility on a 6% constant polyacrylamide gel. By contrast, the two enzymes had identical molecular weights, estimated to be ca 295 kDa on gradient polyacrylamide gel. The involvement of NADP-GDH and GS enzymes in nitrogen assimilation is discussed.     

Role of glutamate dehydrogenase and phosphoenolpyruvate carboxylase activity in ammonium nutrition tolerance in roots

Spinach (Spinacea oleracea L. “Correnta F1”) and pea (Pisum sativum L. “Macrocarpon”) plants were grown in a hydroponic culture with nitrate (5 mM), or ammonium (5 mM) as the nitrogen source. Dry matter accumulation declined dramatically in spinach plants fed with ammonium, whereas there was no change in pea plants when compared with nitrate-fed plants. Data obtained from δ¹⁵N, the organic nitrogen content, N-assimilation enzyme activity, glutamine synthetase (L-glutamate:ammonia-ligase; EC 6.3.1.2), glutamate dehydrogenase (L-glutamate:NAD⁺-oxidoreductase; EC 1.4.1.2) and enzymes from the tricarboxylic acid cycle suggest that ammonium incorporation into organic nitrogen is localized in the roots in pea plants and in the shoots in spinach plants. Distribution of incorporated ammonium (in shoots and roots) may determine ammonium tolerance. Our results show that unlike in spinach plants, in pea plants, an ammonium-tolerant species, GDH enzyme plays an important role in ammonium detoxification by its incorporation into amino acids. Furthermore, phosphoenolpyruvate carboxylase (phosphate:oxaloacetate-carboxy-lyase; EC 4.1.1.31) and pyruvate kinase (ATP:pyruvate-2-O-phosphotransferase; EC 2.7.1.40) activities reflect a major flow of carbon for ammonium assimilation through oxalacetate in pea plants and through pyruvate in spinach plants. The differences in the sensitivity to ammonium between the species are discussed in terms of differences in the site of ammonium assimilation as well as in the nitrogen assimilation ways.     

Purification, properties, and regulation of glutamic dehydrogenase of Bacillus licheniformis

Cell-free extracts of Bacillus licheniformis and B. cereus were found to contain high specific activities of nicotinamide adenine dinucleotide phosphate (NADP)-dependent-l-glutamate dehydrogenase [EC 1.4.1.4; l-glutamate: NADP oxidoreductase (deaminating)]. Maximum specific activities were found in extracts of cells during the late exponential phase of growth when ammonium ion served as the sole source of nitrogen. Extremely low specific activities were detected throughout the growth cycle when l-glutamate or Casamino Acids served as the source of carbon and nitrogen. The enzyme was purified 55-fold from crude extracts of B. licheniformis, and apparent kinetic constants were determined. Sigmoidal saturation kinetics were not observed, and various adenylates had no effect on the enzyme. Repression of enzyme synthesis during growth on l-glutamate or Casamino Acids was partially overcome by additions of glucose or pyruvate, and this apparent derepression was totally abolished by inhibitors of ribonucleic acid and protein synthesis. Similarly, additions of l-glutamate or Casamino Acids to cells growing on glucose-ammonium ion resulted in strong repression of enzyme synthesis. It is suggested that the enzyme serves an anabolic role in metabolism. Nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase activity was not detected in five species of Bacillus, irrespective of nutritional conditions or of the physiological age of cells.     

Purification and properties of NADP-dependent glutamate dehydrogenase from Streptomyces fradiae

Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The Mr of the native enzyme was determined to be 200,000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of Mr 49,000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and L-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9.2 for oxidative deamination of glutamate and 8.4 for reductive amination of 2-oxoglutarate. The Michaelis constants (Km) were 28.6 mM for L-glutamate and 0.12 mM for NADP. Km values for reductive amination were 1.54 mM for 2-oxoglutarate, 0.07 mM for NADPH and 30.8 mM for NH+4. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.     

Sources of NADPH in yeast vary with carbon source

Production of NADPH in Saccharomyces cerevisiae cells grown on glucose has been attributed to glucose-6-phosphate dehydrogenase (Zwf1p) and a cytosolic aldehyde dehydrogenase (Ald6p) (Grabowska, D., and Chelstowska, A. (2003) J. Biol. Chem. 278, 13984-13988). This was based on compensation by overexpression of Ald6p for phenotypes associated with ZWF1 gene disruption and on the apparent lethality resulting from co-disruption of ZWF1 and ALD6 genes. However, we have found that a zwf1Delta ald6Delta mutant can be constructed by mating when tetrads are dissected on plates with a nonfermentable carbon source (lactate), a condition associated with expression of another enzymatic source of NADPH, cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p). We demonstrated previously that a zwf1Delta idp2Delta mutant loses viability when shifted to medium with oleate or acetate as the carbon source, apparently because of the inadequate supply of NADPH for cellular antioxidant systems. In contrast, the zwf1Delta ald6Delta mutant grows as well as the parental strain in similar shifts. In addition, the zwf1Delta ald6Delta mutant grows slowly but does not lose viability when shifted to culture medium with glucose as the carbon source, and the mutant resumes growth when the glucose is exhausted from the medium. Measurements of NADP(H) levels revealed that NADPH may not be rapidly utilized in the zwf1Delta ald6Delta mutant in glucose medium, perhaps because of a reduction in fatty acid synthesis associated with loss of Ald6p. In contrast, levels of NADP+ rise dramatically in the zwf1Delta idp2Delta mutant in acetate medium, suggesting a decrease in production of NADPH reducing equivalents needed both for biosynthesis and for antioxidant functions.     

Glutamate dehydrogenase from Escherichia coli: induction, purification and properties of the enzyme.

When Escherichia coli was grown in a minimum medium with glucose as sole carbon source and a proper level of ammonia, NADP+ specific glutamate dehydrogenase (L-glutamate: NADP+ oxidoreductase (deaminating), ED 1.4.1.4) was induced. The enzyme was solubilized by French press treatment and purified to homogeneity by (NH4)2SO4 fractionation, heat treatment followed by DEAE-cellulose, hydroxylapatite and Bio-Gel chromatography with an overall yield of 30%. The enzyme proved to be heat stable and relatively resistant to protein denaturants. The optimum of enzymic activity for the reductive amination is at pH 8 and at pH 9 for the oxidative deamination. The activity is affected by adenine nucleotides. The molecular weight (about 250 000 for the native form and 46 000 for the inactive subunit) and amino acid composition, suggest strict similarities with the NADP+ enzyme from fungal origin.     

Properties of glutamate dehydrogenase purified from Bacteroides fragilis

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.     

Occurrence of a novel yeast enzyme, L-lysine epsilon-dehydrogenase, which catalyses the first step of lysine catabolism in Candida albicans

The yeast Candida albicans is able to utilize L-lysine as the sole nitrogen and carbon source accompanied by intracellular accumulation of alpha-aminoadipate-delta-semialdehyde. A novel yeast amino acid dehydrogenase catalysing the oxidative deamination of the epsilon-group of L-lysine was found in this yeast. The enzyme, L-lysine epsilon-dehydrogenase, is strongly induced in cells grown on L-lysine as the sole nitrogen source. The enzyme is specific for both L-lysine and NADP+. The Km values were determined to be 0.87 mM for L-lysine and 0.071 mM for NADP+. An apparent Mr of 87,000 was estimated by gel filtration. The enzyme has maximum activity at pH 9.5 and a temperature optimum of 32 degrees C under our assay conditions.     

Regulation of carbon and nitrogen metabolisms in rice roots by 2-oxoglutarate at the level of hexokinase

Feeding experiments were designed, to investigate the role of 2-oxoglutarate (2-OG) in regulation of carbon and nitrogen metabolisms in non-photosynthetic tissues of rice ( Oryza sativa L.), and enzyme activities involved in the metabolisms as well as contents of several relating metabolites were determined in the roots. The enhancement of 2-OG level by feeding 2-OG or metabolizable sugars [sucrose (Suc) or glucose (Glc)], rather than by feeding non-metabolizable carbon sources (mannose or mannitol), led to increase in enzyme activities, including hexokinase (HXK, EC 2.7.1.1), nicotinamide adenine dinucleotide phosphate (NADP)⁺-dependent isocitrate dehydrogenase (NADP⁺-ICDH, EC 1.1.1.42), phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), glutamine synthetase (GS, EC 6.3.1.2) and the reduced form of nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14). In addition, the increase in ammonium uptake, glutamine and glutamate (Glu) as well as the decrease in soluble carbohydrates were observed. The effects of feeding 2-OG or metabolizable sugars were reversed by feeding of N- acetyl-glucosamine (NAG; a HXK inhibitor). The decreased 2-OG level by the feeding of NAG alone led to increase in soluble carbohydrates and decrease in the enzyme activities, ammonium uptake as well as Glu content. The effects of NAG were reversed by supply of 2-OG, Suc and Glc. These results suggest that nitrogen uptake and assimilation as well as their related carbohydrate metabolism in rice roots were regulated in coordination by 2-OG level, and HXK activity was involved in the regulation of 2-OG.     

Nitrogen regulation of arginase in Neurospora crassa.

The final products of the arginine catabolism that can be utilized as a nitrogen source in Neurospora crassa are ammonium, glutamic acid, and glutamine. The effect of these compounds on arginase induction by arginine was studied. In wild-type strain 74-A, induction by arginine was almost completely repressed by glutamic acid plus ammonium, whereas ammonium or glutamic acid alone had only moderate effects. Arginine products of catabolism also repressed arginase induction. A mutant, ure-1, which lacks urease activity, hyperinduced its arginase with arginine as a nitrogen source. The addition of either ammonium or glutamine produced effects similar to those in the wild-type strain. The effect of ammonium on arginase induction is mediated through its conversion into glutamine. This was demonstrated in mutant am-1, which lacks L-glutamate dehydrogenase activity. In this mutant, the effect of glutamic acid was reduced, and, with ammonium, it was completely lost. The addition of glutamine or glutamic acid plus ammonium to this strain decreased by threefold the induction of arginase by arginine. Proline, a final product of arginine catabolism, competitively inhibited arginase activity. This effect and the repression of arginase by glutamine are examples of negative modulation of the first enzyme in a catabolic pathway by its final products.     

GDH3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae.

It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP+-glutamate dehydrogenase (NADP+-GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP+-GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through an alternative pathway. A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome was carried out. In addition to identifying its cognate sequence at chromosome XIV, the search found that GDH1 showed high identity with a previously recognized open reading frame (GDH3) of chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 were obtained. These were strict glutamate auxotrophs. Our results indicate that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired. This is the first example of a microorganism possessing three pathways for glutamate biosynthesis.     

The structural gene for NADP L-glutamate dehydrogenase in Aspergillus nidulans.

A total of 41 mutants lacking NADP L-glutamate dehydrogenase (NADP-GDH) activity have been studied. All the mutations were located at the gdhA locus within 0-1% recombination of gdhAI. Two mutants, gdhAI and gdhA2, out of five examined, produced cross-reacting material which neutralized NADP-GDH anti-serum. The mutant gdhA9 has altered Km values for all five substrates: ammonium, alpha-ketoglutarate, l-glutamate, NADPH and NADP. The mutant gdhA20 had temperature-sensitive growth, abnormal ammonium-regulation characteristics and thermolabile NADP-GDH activity. These results show that gdhA is the structural gene for NADP-GDH.     

Nitrogen and carbon regulation of lignin peroxidase and enzymes of nitrogen metabolism inPhanerochaete chrysosporium

The regulation of nitrogen metabolism pathways was examined inPhanerochaete chrysosporium in relation to the repression of lignin peroxidase by nitrogen or carbon in this fungus. Under conditions of nitrogen derepression,P. chrysosporium synthesizes the amidohydrolases, formamidase (EC 3.5.1.9) and acetamidase (EC 3.5.1.4) and the enzymes of purine catabolism uricase (EC 1.7.3.3), allantoinase (EC 3.5.2.5), and allantoicase (EC 3.5.3.4). Formamidase is repressed to low levels in the presence of ammonium and there is no apparent control of this enzyme by carbon catabolite repression. Although formamide is a nitrogen source, it is not a carbon source forP. chrysosporium. Glutamate totally represses formamidase. Uricase, allantoinase, and allantoicase are also regulated by nitrogen repression but not carbon catabolite repression. Urease is synthesized at similar levels irrespective of the nitrogen or carbon conditions. The sensitivity of uricase, allantoinase, and allantoicase to nitrogen repression is less than that of formamidase. In contrast to formamidase, glutamate is not a more powerful repressor of uricase, allantoinase, and allantoicase compared with ammonium. No pathway-specific induction is required for the synthesis of formamidase, uricase, allantoinase, and allantoicase. Altogether these features indicate that nitrogen metabolism inP. chrysosporium is similar to that inAspergillus nidulans in its regulation, despite the absence of pathway-specific induction of the enzymes examined. These results are consistent with the existence of a regulatory gene mediating nitrogen catabolite repression similar to theA. nidulans areA gene inP. chrysosporium. Although glycerol acts as a nonrepressive carbon source for lignin peroxidase production (except when used at high concentrations), glutamate totally represses lignin peroxidase even in cultures with glycerol. This indicates that carbon regulation and nitrogen regulation of lignin peroxidase may not be separated inP. chrysosporium.     

Analysis of the kinetic mechanism of halophilic NADP-dependent glutamate dehydrogenase

The amination of 2-oxoglutarate catalyzed by NADP-specific glutamate dehydrogenase (EC 1.4.1.4, L-glutamate:NADP+ oxidoreductase (deaminating)) from Halobacterium halobium has been analyzed by initial rate, graphical analysis, and product and competitive inhibition studies. Initial rate and graphical analysis reveal that a B term (representing 2-oxoglutarate) is not statistically necessary for an initial rate equation. However, the absence of a B term does not distinguish between ordered and random binding of NADPH and ammonia. The patterns of product inhibition by NADP+ and L-glutamate, and competitive inhibition by hydroxylamine and succinate permit deduction of the kinetic mechanism as ordered, with NADPH, 2-oxoglutarate and ammonia added in that order, and L-glutamate release preceding NADP+ release.