Evaluating the Contribution of Glutamate Dehydrogenase and the Glutamate Synthase Cycle to Ammonia Assimilation by Four Ectomycorrhizal Fungal Isolates

1996 ◽  
Vol 23 (2) ◽  
pp. 151 ◽  
Author(s):  
MH Turnbull ◽  
R Goodall ◽  
GR Stewart
Keyword(s):  
Glutamate Dehydrogenase ◽  
Glutamate Synthase ◽  
Ammonium Assimilation ◽  
Major Product ◽  
Ammonia Assimilation ◽  
Gas Chromatography Mass Spectrometry ◽  
Methionine Sulfoximine ◽  
Fungal Isolates ◽  
Glutamine Synthesis ◽  
Fungal Tissue

Combined gas chromatography-mass spectrometry were used to evaluate the contributions of glutamate dehydrogenase (GDH) and the glutamate synthase cycle in 15N-labelled ammonium assimilation by four ectomycorrhizal fungal isolates. In all four species (Elaphomyces, Amanita, Pisolithus and Gautieria), glutamine was the major product accumulated following transfer of 14-day-old nitrogen-limited cultures to fresh medium. Label was rapidly assimilated into fungal tissue, with rates of 733 nmol g-1 FW h-1 in Pisolithus, 972 nmol g-1 FW h-1 in Amanita, 2760 nmol g-1 FW h-1 in Gautieria and 6756 nmol g-1 FW h-1 in Elaphomyces sp in the first 4 h of incubation. Incorporation of [15N]ammonium was sensitive to the inhibitory effects of both methionine sulfoximine (MSX, an inhibitor of glutamine synthetase (GS)) and albizziin (an inhibitor of glutamate synthase (GOGAT)) in three species (Amanita, Gautieria and Pisolithus) and labelling patterns were consistent with the action of the glutamate synthase cycle in ammonium assimilation. In all three species glutamine synthesis was almost totally blocked by MSX and there was no continued incorporation of 15N into glutamate. Elaphomyces displayed high levels of total incorporation of labelled ammonium in mycelium even in the presence of MSX, although incorporation into glutamine was reduced by 88%. This inhibition of GS by MSX, in addition to its partial inhibition by albizziin suggests strongly the action of glutamate synthase cycle in ammonium assimilation. The reduction in label entering glutamate under the influence of albizziin is direct evidence for the inhibition of GOGAT activity. However, MSX treatment had the effect of increasing significantly the quantity of label recovered in both glutamate and alanine. In the absence of GS inhibition there is clearly competition for ammonium which under normal physiological conditions results in assimilation through the glutamate synthase cycle. However, when GS is blocked by MSX label is able to cycle through the GDH pathway. Extra keywords: ectomycorrhiza, ammonium assimilation, glutamate synthase cycle, glutamate dehydrogenase, amino acid metabolism.

scholarly journals The Presence of Glutamate Dehydrogenase Is a Selective Advantage for the Cyanobacterium Synechocystis sp. Strain PCC 6803 under Nonexponential Growth Conditions

1999 ◽  
Vol 181 (3) ◽  
pp. 808-813 ◽  
Author(s):  
S. Chávez ◽  
J. M. Lucena ◽  
J. C. Reyes ◽  
F. J. Florencio ◽  
P. Candau
Keyword(s):  
Glutamate Dehydrogenase ◽  
Glutamate Synthase ◽  
Ammonium Assimilation ◽  
Selective Advantage ◽  
Growth Conditions ◽  
Null Mutant ◽  
Wild Type ◽  
Unicellular Cyanobacterium ◽  
Glutamate Synthesis ◽  
Pcc 6803

ABSTRACT The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 has two putative pathways for ammonium assimilation: the glutamine synthetase-glutamate synthase cycle, which is the main one and is finely regulated by the nitrogen source; and a high NADP-dependent glutamate dehydrogenase activity (NADP-GDH) whose contribution to glutamate synthesis is uncertain. To investigate the role of the latter, we used two engineered mutants, one lacking and another overproducing NADP-GDH. No major disturbances in the regulation of nitrogen-assimilating enzymes or in amino acids pools were detected in the null mutant, but phycobiline content, a sensitive indicator of the nutritional state of cyanobacterial cells, was significantly reduced, indicating that NADP-GDH plays an auxiliary role in ammonium assimilation. This effect was already prominent in the initial phase of growth, although differences in growth rate between the wild type and the mutants were observed at this stage only at low light intensities. However, the null mutant was unable to sustain growth at the late stage of the culture at the point when the wild type showed the maximum NADP-GDH activity, and died faster in ammonium-containing medium. Overexpression of NADP-GDH improved culture proliferation under moderate ammonium concentrations. Competition experiments between the wild type and the null mutant confirmed that the presence of NADP-GDH confers a selective advantage toSynechocystis sp. strain PCC 6803 in late stages of growth.

scholarly journals Metabolic Engineering of Ammonium Assimilation in Xylose-Fermenting Saccharomyces cerevisiae Improves Ethanol Production

2003 ◽  
Vol 69 (8) ◽  
pp. 4732-4736 ◽  
Author(s):  
Christophe Roca ◽  
Jens Nielsen ◽  
Lisbeth Olsson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Dilution Rate ◽  
Glutamate Dehydrogenase ◽  
Ethanol Yield ◽  
Continuous Cultivation ◽  
Ammonium Assimilation ◽  
Batch Cultivation ◽  
Ammonia Assimilation ◽  
Xylose Utilization

ABSTRACT Cofactor imbalance impedes xylose assimilation in Saccharomyces cerevisiae that has been metabolically engineered for xylose utilization. To improve cofactor use, we modified ammonia assimilation in recombinant S. cerevisiae by deleting GDH1, which encodes an NADPH-dependent glutamate dehydrogenase, and by overexpressing either GDH2, which encodes an NADH-dependent glutamate dehydrogenase, or GLT1 and GLN1, which encode the GS-GOGAT complex. Overexpression of GDH2 increased ethanol yield from 0.43 to 0.51 mol of carbon (Cmol) Cmol−1, mainly by reducing xylitol excretion by 44%. Overexpression of the GS-GOGAT complex did not improve conversion of xylose to ethanol during batch cultivation, but it increased ethanol yield by 16% in carbon-limited continuous cultivation at a low dilution rate.

Effects of glutamine deprivation on protein synthesis in a model of human enterocytes in culture

2001 ◽  
Vol 281 (6) ◽  
pp. G1340-G1347 ◽  
Author(s):  
Olivier Le Bacquer ◽  
Hassan Nazih ◽  
Hervé Blottière ◽  
Dominique Meynial-Denis ◽  
Christian Laboisse ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
De Novo ◽  
Gas Chromatography Mass Spectrometry ◽  
Cell Protein ◽  
Methionine Sulfoximine ◽  
Synthesis Rate ◽  
Free Medium ◽  
Glutamine Deprivation ◽  
Glutamine Synthesis

To assess the effect of glutamine availability on rates of protein synthesis in human enterocytes, Caco-2 cells were grown until differentiation and then submitted to glutamine deprivation produced by exposure to glutamine-free medium or methionine sulfoximine [l- S-[3-amino-3-carboxypropyl]- S-methylsulfoximine (MSO)], a glutamine synthetase inhibitor. Cells were then incubated with2H3-labeled leucine with or without glutamine, and the fractional synthesis rate (FSR) of total cell protein was determined from2H3-labeled enrichments in protein-bound and intracellular free leucine measured by gas chromatography-mass spectrometry. Both protein FSR (28 ± 1.5%/day) and intracellular glutamine concentration (6.1 ± 0.6 μmol/g protein) remained unaltered when cells were grown in glutamine-free medium. In contrast, MSO treatment resulted in a dramatic reduction in protein synthesis (4.6 ± 0.6 vs. 20.2 ± 0.8%/day, P < 0.01). Supplementation with 0.5–2 mM glutamine for 4 h after MSO incubation, but not with glycine nor glutamate, restored protein FSR to control values (24 ± 1%/day). These results demonstrate that in Caco-2 cells, 1) de novo glutamine synthesis is highly active, since it can maintain intracellular glutamine pool during glutamine deprivation, 2) inhibition of glutamine synthesis is associated with reduced protein synthesis, and 3) when glutamine synthesis is depressed, exogenous glutamine restores normal intestinal FSR. Due to the limitations intrinsic to the use of a cell line as an experimental model, the physiological relevance of these findings for the human intestine in vivo remains to be determined.

Enzymes of ammonia assimilation in Rhizobium leguminosarum bacteroids

1975 ◽  
Vol 21 (7) ◽  
pp. 1009-1012 ◽  
Author(s):  
W. G. W. Kurz ◽  
D. A. Rokosh ◽  
T. A. Larue
Keyword(s):  
Glutamine Synthetase ◽  
Glutamate Dehydrogenase ◽  
Rhizobium Leguminosarum ◽  
Glutamate Synthase ◽  
Dinitrogen Fixation ◽  
Ammonia Assimilation ◽  
Enzymes Of Ammonia Assimilation

The activities of the following enzymes were studied in connection with dinitrogen fixation in pea bacteroids: glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming)) (EC 6.3.1.2) (GS); glutamate dehydrogenase (NADP+) (L-glutamate:NADP+ oxidoreductase (deaminating)) (EC 1.4.1.4) (GDH); glutamate synthase (L-glutamine:2-oxoglutarate aminotransferase (NADPH-oxidizing)) (EC 2.6.1.53) (GOGAT). GS activity was high throughout the growth of the plant and GOGAT activity was always low. It is unlikely that GDH or the GS–GOGAT pathway can account for the incorporation of ammonia from dinitrogen fixation in the pea bacteroid.

Enzymes of Nitrogen Metabolism in Legume Nodules: a Comparative Study

1978 ◽  
Vol 5 (5) ◽  
pp. 553 ◽  
Author(s):  
MJ Boland ◽  
HM Fordyce ◽  
RM Greenwood
Keyword(s):  
Glutamine Synthetase ◽  
Comparative Study ◽  
Glutamate Dehydrogenase ◽  
Nitrogen Metabolism ◽  
Glutamate Synthase ◽  
Ammonia Assimilation ◽  
Legume Species

Levels of activity of glutamine synthetase, glutamate dehydrogenase and NADH-dependent glutamate synthase in nodule cytoplasm extracts of twelve herbaceous legume species have been measured. Nodules of all species contained substantial quantities of glutamine synthetase. Levels of glutamate synthase were found to be between 7 and 100% of those of glutamine synthetase, while levels of glutamate dehydrogenase varied widely between 0.2 and 150% of those of glutamine synthetase. The estimated Km for hydroxylamine of glutamine synthetase was found to vary between 0.02 and 0.5 mM in nine species tested, while that of glutamate dehydrogenase for ammonia varied between 0.03 M and 0.1 M in the four species containing significant levels of that enzyme. It is proposed that the pathway of ammonia assimilation via glutamine synthetase and NADH-dependent glutamate synthase-catalysed reactions is universal in legume nodule metabolism.

Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.)

Planta ◽  
2005 ◽  
Vol 222 (4) ◽  
pp. 667-677 ◽  
Author(s):  
Magali Feraud ◽  
Céline Masclaux-Daubresse ◽  
Sylvie Ferrario-Méry ◽  
Karine Pageau ◽  
Maud Lelandais ◽  
...  
Keyword(s):  
Nicotiana Tabacum ◽  
Glutamate Synthase ◽  
Ammonium Assimilation ◽  
Vascular Cells ◽  
Nicotiana Tabacum L

Properties of NAD-dependent glutamate dehydrogenase from the tylosin producer Streptomyces fradiae

1997 ◽  
Vol 43 (11) ◽  
pp. 1005-1010 ◽  
Author(s):  
Kien Trung Nguyen ◽  
Lieu Thi Nguyen ◽  
Jan Kopecký ◽  
Vladislav Běhal
Keyword(s):  
Key Words ◽  
Glutamate Dehydrogenase ◽  
Reductive Amination ◽  
Divalent Cations ◽  
Ammonium Assimilation ◽  
Alkaline Ph ◽  
Streptomyces Fradiae ◽  
Oxidative Deamination

Glutamate dehydrogenase is an enzyme responsible for ammonium assimilation and glutamate catabolism in organisms. The tylosin producer Streptomyces fradiae possesses both NADP- and NAD-dependent glutamate dehydrogenases. The latter enzyme was purified 498-fold with a 7.5% recovery by a six-step protocol. The enzyme is composed of two subunits, each of Mr 47 000, and could form active aggregates of four or eight subunits. Its activity was inactivated by alkaline pH or temperatures of −20 °C or above 40 °C. Activities assayed in the direction of oxidative deamination and reductive amination were optimal at pH 9.2 and 8.8, respectively, and at temperatures of 30–35 °C. No activity was found when NAD(H) was replaced with NADP(H). The Km values were 32.2 mM for L-glutamate, 0.3 mM for NAD+, 3.4 mM for 2-ketoglutarate, 14.2 mM for NH4+, and 0.05 mM for NADH. Deamination activity was partially inhibited by adenyl nucleotides and several divalent cations; amination activity was not affected by the nucleotides but significantly inhibited by Cu2+ or Ni2+.Key words: Streptomyces fradiae, NAD-dependent glutamate dehydrogenase, purification, properties.

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