Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes: studies with the continuous-culture technique.

Ammonia-nitrogen-limited continuous cultures of Escherichia coli and Klebsiella aerogenes contain induced levels of glutamine synthetase that is deadenylyated (i.e., fully active). In the presence of excess ammonia or glutamate in glucose-limited cultures of E. coli, glutamine synthetase is repressed and adenylylated (inactive). The average state of adenylylation (n) is a linear function of the specific growth rate. At low specific growth rates, glutamine synthetase is adenylylated; as the specific growth rate increases, n decreases, approaching 0 to 2 at rapid growth rates. The average state of adenylylation correlates well with the intracellular concentrations and ratios of alpha-ketoglutarate and glutamine, which are key effectors in the adenylylation-deadenylylation systems. E. coli and K. aerogenes differ markedly in their growth yields, growth rates, and enzymatic composition during nitrogen limitation. The data suggest that, unlike K. aerogenes, E. coli W uses glutamate dehydrogenase to incorporate ammonia during nitrogen limitation. In E. coli, glutamate dehydrogenase is progressively induced during nitrogen limitation when mu (growth rate) approaches mumax. In contrast, in K. aerogenes glutamate dehydrogenase is repressed during nitrogen limitation, whereas glutamate synthase, an alternative supplier of glutamate to the cell, is induced. Data are presented that support the regulatory schemes proposed for the control of glutamine synthetase activity by induction-repression phenomena and adenylylation-deadenylylation reaction. We propose that the intracellular ratio of alpha-ketoglutarate to glutamine may be the most important physiological parameter in determining the activity of glutamine synthetase.     

The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms.

In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on ammonium sulfate as a nitrogen source, (ii) Northern (RNA) blot analysis shows that the steady-state level of GDH1 mRNA is strongly lowered in a hap2 mutant, (iii) expression of a GDH1-lacZ fusion is drastically reduced in hap mutants, (iv) NADP-GDH activity is several times lower in the hap mutants compared with that in the isogenic wild-type strain, and finally, (v) site-directed mutagenesis of two consensual HAP binding sites in the GDH1 promoter strongly reduces expression of GDH1 and makes it HAP independent. Expression of GDH1 is also regulated by the carbon source, i.e., expression is higher on lactate than on ethanol, glycerol, or galactose, with the lowest expression being found on glucose. Finally, we show that a hap2 mutation does not affect expression of other genes involved in nitrogen metabolism (GDH2, GLN1, and GLN3 encoding, respectively, the NAD-GDH, glutamine synthetase, and a general activator of several nitrogen catabolic genes). The HAP complex is known to regulate expression of several genes involved in carbon metabolism; its role in the control of GDH1 gene expression, therefore, provides evidence for a cross-pathway regulation between carbon and nitrogen metabolisms.     

Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress

The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. (15)NH(4) labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized (15)N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while (15)N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt.     

浑球红假单胞菌氨同化途径的研究

本文测定了浑球红假单胞菌(Rhodobacter sphaeroides)菌株601谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、谷氨酸脱氢酶(GDH)和丙氨酸脱氢酶(ADH)的活性。低氨时,GS/GOGAT活力高,GDH活力低,高氨时,GS/GOGAT活力低,GDH活力高。在以分子氮或低浓度氨为氮源的培养条件下,加入GS抑制刑MSX(L—methionine—DL—sulphoximine),细菌生长受到抑制。但是,生长在以谷氨酸为氮源的细菌则不受影响。上述结果表明,浑球红假单胞菌菌株601氨同化是通过GS/GOGAT途径和GDH途径。     

An NAD(+)-dependent glutamate dehydrogenase cloned from the ruminal ciliate protozoan, Entodinium caudatum

An NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.24) was cloned from the ruminal ciliate protozoan, Entodinium caudatum. The gene had high sequence similarity to GDH genes from the Bacteroides (class)--a class of bacteria which is highly represented in the rumen. When expressed in Escherichia coli the enzyme had a high affinity for ammonia and alpha-ketoglutarate (apparent K(m) of 2.33 and 0.71 mM, respectively) and a low affinity for glutamate (apparent K(m) of 98 mM). GDH activity and GDH mRNA concentration were increased by incubating washed E. caudatum cells with ammonia and antibiotics. These results suggest that the GDH is an anabolic enzyme catalysing the assimilation of ammonia by E. caudatum in the rumen and that the gene was probably acquired by lateral gene transfer from a ruminal bacterium.     

Ammonia assimilation and glutamate formation in the anaerobe Selenomonas ruminantium.

Selenomonas ruminantium was found to possess two pathways for NH4+ assimilation that resulted in net glutamate synthesis. One pathway fixed NH4+ through the action of an NADPH-linked glutamate dehydrogenase (GDH). Maximal GDH activity required KCl (about 0.48 M), but a variety of monovalent salts could replace KCl. Complete substrate saturation of the enzyme by NH4+ did not occur, and apparent Km values of 6.7 and 23 mM were estimated. Also, an NADH-linked GDH activity was observed but was not stimulated by KCl. Cells grown in media containing non-growth-rate-limiting concentrations of NH4+ had the highest levels of GDH activity. The second pathway fixed NH4+ into the amide of glutamine by an ATP-dependent glutamine synthetase (GS). The GS did not display gamma-glutamyl transferase activity, and no evidence for an adenylylation/deadenylylation control mechanism was detected. GS activity was highest in cells grown under nitrogen limitation. Net glutamate synthesis from glutamine was effected by glutamate synthase activity (GOGAT). The GOGAT activity was reductant dependent, and maximal activity occurred with dithionite-reduced methyl viologen as the source of electrons, although NADPH or NADH could partially replace this artificial donor system. Flavin adenine dinucleotide, flavin mononucleotide, or ferredoxin could not replace methyl viologen. GOGAT activity was maximal in cells grown with NH4+ as sole nitrogen source and decreased in media containing Casamino Acids.     

Glutamate dehydrogenases from tissues of the ribbed mussel Modiolus demissus: ADP activation and possible physiological significance.

Glutamate dehydrogenase (E.C. 14.1.3) was localized in the mitochondria from heart, gill, mantle and hepatopancreas of this euryhaline bivalve mollusc. Activity levels were low (0.1-0.4 mumoles/min/g wet weight) in all tissues when assayed in the glutamate forming direction. Partially purified gill mitochondrial GDH was most active at pH 8.5. The rate in the glutamate deaminating direction was 10-20% of the rate in the glutamate forming direction. ADP at apparent Ka concentrations of micrometer (glutamate formation) and 170 micrometer (glutamate deamination) enhanced GDH activity, 8- and 4-fold respectively. GDH, in vivo, is probably in the activated form and appears to function in glutamate synthesis rather than ammonia formation. However, based on the low activities obtained, the role of GDH in salinity induced amino acid synthesis seems minimal.     

Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae.

We cloned GDH2, the gene that encodes the NAD-linked glutamate dehydrogenase in the yeast Saccharomyces cerevisiae, by purifying the enzyme, making polyclonal antibodies to it, and using the antibodies to screen a lambda gt11 yeast genomic library. A yeast strain with a deletion-disruption allele of GDH2 which replaced the wild-type gene grew very poorly with glutamate as a nitrogen source, but growth improved significantly when the strain was also provided with adenine or other nitrogenous compounds whose biosynthesis requires glutamine. Our results indicate that the NAD-linked glutamate dehydrogenase catalyzes the major, but not sole, pathway for generation of ammonia from glutamate. We also isolated yeast mutants that lacked glutamate synthase activity and present evidence which shows that normally NAD-linked glutamate dehydrogenase is not involved in glutamate biosynthesis, but that if the enzyme is overexpressed, it may function reversibly in intact cells.     

Induction of glutamate dehydrogenase in the ovine fetal liver by dexamethasone infusion during late gestation

Glucocorticoids near term are known to upregulate many important enzyme systems prior to birth. Glutamate dehydrogenase (GDH) is a mitochondrial enzyme that catalyzes both the reversible conversion of ammonium nitrogen into organic nitrogen (glutamate production) and the oxidative deamination of glutamate resulting in 2-oxoglutarate. The activity of this enzyme is considered to be of major importance in the development of catabolic conditions leading to gluconeogenesis prior to birth. Ovine hepatic GDH mRNA expression and activity were determined in near-term (130 days of gestation, term 147 +/- 4 days) control and acutely dexamethasone-treated (0.07 mg(-1) hr(-1) for 26 hr) fetuses. Dexamethasone infusion had no effect on placental or fetal liver weights. Dexamethasone infusion for 26 hr significantly increased hepatic GDH mRNA expression. This increased GDH mRNA expression was accompanied by an increase in hepatic mitochondrial GDH activity, from 30.0 +/- 7.4 to 58.2 +/- 8.1 U GDH/U CS (citrate synthase), and there was a significant correlation between GDH mRNA expression and GDH activity. The generated ovine GDH sequence displayed significant similarity with published human, rat, and murine GDH sequence. These data are consistent with the in vivo studies that have shown a redirection of glutamine carbon away from net hepatic glutamate release and into the citric acid cycle through the forward reaction catalyzed by GDH, i.e., glutamate to oxoglutarate.     

The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent K _m values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent K _m values were 0.1 mM for -ketoglutarate and 0.22 mM for glutamine.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase     

Nitrogen Metabolism of Lemna minor. II. Enzymes of Nitrate Assimilation and Some Aspects of Their Regulation

In L. minor grown in sterile culture, the primary enzymes of nitrate assimilation, nitrate reductase (NR), nitrite reductase (NiR) and glutamate dehydrogenase (GDH) change in response to nitrogen source. NR and NiR levels are low when grown on amino acids (hydrolyzed casein) or ammonia; both enzymes are rapidly induced on addition of nitrate, while addition of nitrite induces NiR only. Ammonia represses the nitrate induced synthesis of both NR and NiR.NADH dependent GDH activity is low when grown on amino acids and high when grown on nitrate or ammonia, but the activities of NADPH dependent GDH and Alanine dehydro-genase (AIDH) are much less affected by nitrogen source. NADH-GDH and AIDH are induced by ammonia, and it is suggested that these enzymes are involved in primary nitrogen assimilation.     

Growth of Bacillus fastidiosus on glycerol and the enzymes of ammonia assimilation

Bacillus fastidiosus was able to grow on glycerol as a carbon source when allantoin or urate was used as nitrogen source. The primary assimilatory enzyme for glycerol was glycerol kinase; glycerol dehydrogenase could not be detected. The glycerol kinase activity was increased 30-fold in allantoin/glycerol-grown cells as compared to alantoin-grown cells. Under both growth conditions high levels of glutamate dehydrogenase were found. Glutamine synthetase and glutamate synthase activities could not be demonstrated, while low levels of alanine dehydrogenase were present. It is concluded that B. fastidiosus assimilates ammonia by the NADP-dependent glutamate dehydrogenase.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase     

Growth of Pseudomonas aeruginosa mutants lacking glutamate synthase activity.

Mutant strains SU1, SU4, and US1 lacking glutamate synthase (GOGAT) activity were isolated from strains of P. aeruginosa for which histidine is a growth rate-limiting source of nitrogen. Strains SU1 and SU4 were unable to grow when a low concentration of ammonia and a variety of compounds, including histidine, were supplied as sole sources of nitrogen. A revertant of strain SU1, strain 39, produced no GOGAT but high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase and had restored ability to grow on a limited number of nitrogen sources. Strain US1 grew at the same rate in histidine medium as did its parent; it was derepressed for glutamine synthase synthesis, and histidase was less sensitive to repression by ammonia than in the parent strain. We conclude that GOGAT is not essential for growth on histidine but high levels of glutamine synthase are required nd high levels of nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase can sustain growth at low concentrations of ammonia in the absence of GOGAT.     

Enzymes of Ammonia Assimilation, Photosynthesis, and Respiration in Alfalfa Leaves of Different Ages

The activities of enzymes involved in ammonia metabolism ferredoxin-dependent glutamate synthase (Fd-GOGAT), glutamine synthetase (GS) and glutamate dehydrogenase (GDH), the rates of photosynthetic oxygen evolution, dark respiration, and the activity of RuBP carboxylase (RuBPC) were determined in alfalfa (Medicago sativa L.) leaves taken from the apex (apical leaves), from the second to the fourth internode (mature leaves) and from the bottom of the canopy (basal leaves). Photosynthetic rate and the activities of RuBPC, GS and Fd-GOGAT showed their maximum in the mature leaves. The respiration rate together with amino acid and ammonium contents decreased with leaf age, whereas the opposite was true for GDH activity. Basal leaves still maintained substantial levels of chlorophylls, GS and Fd-GOGAT activities and oxygen evolution rate, thus suggesting that photosynthesis has some role in the reassimilation of the nitrogen liberated during protein degradation. This revised version was published online in June 2006 with corrections to the Cover Date.     

不同浓度的NO^—3和NH^＋4对小麦根谷氨酰胺合成酶及其相关酶的影响

利用酶活性测定和 Northern分子杂交等技术 ,研究了小麦幼苗根在不同浓度的 Na NO3 和(NH4) 2 SO4的供应下 ,其谷氨酰胺合成酶 (GS)、天冬酰胺合成酶 (AS)、谷氨酸脱氢酶 (GDH)、硝酸还原酶 (NR)以及 GS- m RNA的变化。结果表明 :NH 4 处理的小麦 ,其根部 GS活性比 NO-3 处理的高 ;高浓度处理的比低浓度处理的高 ;Northern杂交结果说明 GS- m RNA转录量与 GS活性一致 ;3mmol/ L NO-3促进了 AS的活性。AS酶活性变化与 GS酶活性变化无明显依赖关系。在实验的条件下 ,没能测出 GDH的活性 ,不同浓度的 NO-3 和 NH 4 处理对 NR活性没有明显的规律。     

The Role of Glutamine Synthetase and Glutamate Dehydrogenase in Cerebral Ammonia Homeostasis

In the brain, glutamine synthetase (GS), which is located predominantly in astrocytes, is largely responsible for the removal of both blood-derived and metabolically generated ammonia. Thus, studies with [¹³N]ammonia have shown that about 25?% of blood-derived ammonia is removed in a single pass through the rat brain and that this ammonia is incorporated primarily into glutamine (amide) in astrocytes. Major pathways for cerebral ammonia generation include the glutaminase reaction and the glutamate dehydrogenase (GDH) reaction. The equilibrium position of the GDH-catalyzed reaction in vitro favors reductive amination of α-ketoglutarate at pH 7.4. Nevertheless, only a small amount of label derived from [¹³N]ammonia in rat brain is incorporated into glutamate and the α-amine of glutamine in vivo. Most likely the cerebral GDH reaction is drawn normally in the direction of glutamate oxidation (ammonia production) by rapid removal of ammonia as glutamine. Linkage of glutamate/α-ketoglutarate-utilizing aminotransferases with the GDH reaction channels excess amino acid nitrogen toward ammonia for glutamine synthesis. At high ammonia levels and/or when GS is inhibited the GDH reaction coupled with glutamate/α-ketoglutarate-linked aminotransferases may, however, promote the flow of ammonia nitrogen toward synthesis of amino acids. Preliminary evidence suggests an important role for the purine nucleotide cycle (PNC) as an additional source of ammonia in neurons (Net reaction: l-Aspartate?+?GTP?+?H₂O?→?Fumarate?+?GDP?+?P_i?+?NH₃) and in the beat cycle of ependyma cilia. The link of the PNC to aminotransferases and GDH/GS and its role in cerebral nitrogen metabolism under both normal and pathological (e.g. hyperammonemic encephalopathy) conditions should be a productive area for future research.     

Occurrence of glutamate dehydrogenase isoenzymes during growth of Oocystis alga

Oocystis sp., a unicellular green alga, contained two glutamate dehydrogenase isoenzymes: one was specific for NADH and the other for NADPH. Activity staining after gel electrophoresis indicated that one component in NADH-GDH was not specific for the cofactor and three components in NADPH-GDH. The optimal concentration of substrate, purification procedure and kinetic properties of both glutamate dehydrogenase (GDH) enzymes in vitro are presented. The kinetics of growth, nutrient removal and enzyme activities for Oocystis growing in wastewater showed that ammonia was preferentially utilized over nitrate and the medium was depleted before the maximum population was obtained in indoor culture. There was a sharp increase in NADPH-GDH activity following the exhaustion of ammonia from the medium but NADH-GDH activity remained unchanged. The NADPH-GDH activity at the outset increased exponentially with time in greenhouse culture but then decreased sharply accompained by a rapid increase in biomass and nitrite concentration. The K(m) values for ammonia in this algal GDH was high, while glutamate synthase activity was not detected; this suggests that Oocystis may adapt to conditions of ammonia limitation by producing large quantities of NADPH-GDH instead of using glutamate synthase pathway.     

Glutamate synthesis in Streptomyces coelicolor.

Both glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) are involved in glutamate synthesis in Streptomyces coelicolor. The highest levels of GDH were seen in extracts of cells grown with high levels of ammonium as the nitrogen source. GOGAT activity was reduced two- to threefold in extracts of cells grown with good sources of glutamate. S. coelicolor mutants deficient in GOGAT (Glt-) required glutamate for growth with L-alanine, asparagine, arginine, or histidine as the nitrogen source but grew like wild-type cells when ammonium, glutamine, or aspartate was the nitrogen source. The glt mutations were tightly linked to hisA1. Mutants deficient in both GOGAT and GDH (Gdh-) required glutamate for growth in all media. The gdh-5 mutation was mapped to the left region of the S. coelicolor chromosomal map, between proA1 and uraA1.