Glutamine Synthetase-glutamate Synthase Pathway Research Articles

Atmospheric NO2 enhances tolerance to low temperature by promoting nitrogen and carbon metabolism in tobacco

With abnormal global climate change and the frequent occurrence of extreme weather, low-temperature stress poses an increasingly serious threat to the diversity of plants. Low temperatures accompanied by limitations in nitrogen cause a series of morphological, physiological, and molecular changes in plants. Nitrogen dioxide (NO2) is a gas that is considered to be a toxic air pollutant. However, NO2 in the atmosphere can be absorbed by plants and participate in nitrogen metabolism. In this study, tobacco (Nicotiana tabacum L.) seedlings were fumigated with a concentration of 4µL·L-1 NO2 at 4 ℃ for 10 days. NO2 promoted the glutamine synthetase-glutamate synthase (GS/GOGAT) pathway in the leaves at low temperatures, which transforms more organic nitrogen that can be directly utilized by the plants, and improves the skeleton for carbon metabolism. Moreover, gamma-aminobutyric acid (GABA) was generated through the ornithine and glutamate pathways, and the biosynthesis of proline was also enhanced after treatment with NO2. Together, these compounds regulate the osmotic balance of tobacco leaf cells under low-temperature stress. NO2 activated the ascorbate-glutathione (AsA-GSH) cycle in the leaves under low-temperature stress, and this antioxidant enzyme system synergistically removed the intracellular free radicals that result from reactive oxygen species. Additionally, NO2 significantly increased the content of nitric oxide (NO) in the leaves under low-temperature stress and enhanced the opening of stomata, thus, improving photosynthesis in the leaves. The biosynthesis of chlorophyll in the leaves was inhibited by low-temperature stress, but NO2 promotes the biosynthesis of chlorophyll directly or through the nitric acid signaling pathway and improves the ability of plants to capture light energy. NO2 also alleviates the photoinhibition induced by cold stress by regulating photosynthetic electron transfer and absorbing more energy for the assimilation of photosynthetic carbon. This improves the photochemical efficiency of tobacco leaves. In conclusion, NO2 enhanced the tolerance of tobacco seedlings to low temperature by regulating nitrogen metabolism, the osmotic balance, antioxidant system, and photosynthesis.

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides.

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.

Metabolic regulation of Escherichia coli and its glnG and zwf mutants under nitrogen limitation

The effect of nitrogen (N)-limitation on the metabolism of the wild type Escherichia coli BW25113, its glnG, and zwf mutants was investigated based on the chemostat cultures at the dilution rate of 0.2 h −1 under both carbon (C)-limitation and N-limitation. It was shown that gdhA gene expression was down-regulated, and glutamine synthetase-glutamate synthase (GS/GOGAT) pathway was mainly utilized under N-limitation in E. coli BW25113. It was also shown that the specific glucose consumption rate and the specific CO 2 evolution rate (CER) were higher in glnG mutant as compared to the wild type under C-limitation. It was also shown that GOGAT is the main pathway for N assimilation under C-limitation, while GDH is the main pathway under N-limitation in glnG mutant. In zwf mutant, glnA, glnG, and gltD gene expressions were significantly down-regulated, while malic enzyme was significantly activated for NADPH production under both C-limitation and N-limitation.

Physiological and Transcriptomic Aspects of Urea Uptake and Assimilation in Arabidopsis Plants

Urea is the major nitrogen (N) form supplied as fertilizer in agriculture, but it is also an important N metabolite in plants. Urea transport and assimilation were investigated in Arabidopsis (Arabidopsis thaliana). Uptake studies using (15)N-labeled urea demonstrated the capacity of Arabidopsis to absorb urea and that the urea uptake was regulated by the initial N status of the plants. Urea uptake was stimulated by urea but was reduced by the presence of ammonium nitrate in the growth medium. N deficiency in plants did not affect urea uptake. Urea exerted a repressive effect on nitrate influx, whereas urea enhanced ammonium uptake. The use of [(15)N]urea and [(15)N]ammonium tracers allowed us to show that urea and ammonium assimilation pathways were similar. Finally, urea uptake was less efficient than nitrate uptake, and urea grown-plants presented signs of N starvation. We also report the first analysis, to our knowledge, of Arabidopsis gene expression profiling in response to urea. Our transcriptomic approach revealed that nitrate and ammonium transporters were transcriptionally regulated by urea as well as key enzymes of the glutamine synthetase-glutamate synthase pathway. AtDUR3, a high-affinity urea transporter in Arabidopsis, was strongly up-regulated by urea. Moreover, our transcriptomic data suggest that other genes are also involved in urea influx.

Glutamine Synthetase-Glutamate Synthase Pathway and Glutamate Dehydrogenase Play Distinct Roles in the Sink-Source Nitrogen Cycle in Tobacco

Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.

Pathway choice in glutamate synthesis in Escherichia coli.

Escherichia coli has two primary pathways for glutamate synthesis. The glutamine synthetase-glutamate synthase (GOGAT) pathway is essential for synthesis at low ammonium concentration and for regulation of the glutamine pool. The glutamate dehydrogenase (GDH) pathway is important during glucose-limited growth. It has been hypothesized that GDH is favored when the organism is stressed for energy, because the enzyme does not use ATP as does the GOGAT pathway. The results of competition experiments between the wild-type and a GDH-deficient mutant during glucose-limited growth in the presence of the nonmetabolizable glucose analog alpha-methylglucoside were consistent with the hypothesis. Enzyme measurements showed that levels of the enzymes of the glutamate pathways dropped as the organism passed from unrestricted to glucose-restricted growth. However, other conditions influencing pathway choice had no substantial effect on enzyme levels. Therefore, substrate availability and/or modulation of enzyme activity are likely to be major determinants of pathway choice in glutamate synthesis.

Dephosphorylation of the phosphoprotein P(II) in Synechococcus PCC 7942: identification of an ATP and 2-oxoglutarate-regulated phosphatase activity.

The phosphorylation state of the putative signal transduction protein P(II) from the cyanobacterium Synechococcus sp. strain PCC 7942 depends on the cellular state of nitrogen and carbon assimilation. In this study, dephosphorylation of phosphorylated P(II) protein (P[II]-P) was investigated both in vivo and in vitro. The in vivo studies implied that P(II)-P dephosphorylation is regulated by inhibitory metabolites involved in the glutamine synthetase-glutamate synthase pathway of ammonium assimilation. An in vitro assay for P(II)-P dephosphorylation was established that revealed a Mg2+-dependent P(II)-P phosphatase activity. P(II)-P phosphatase and P(II) kinase activities could be separated biochemically. A partially purified P(II)-P phosphatase preparation also catalysed the dephosphorylation of phosphoserine/phosphothreonine residues on other proteins in a Mg2+-dependent manner. However, only dephosphorylation of P(II)-P was regulated by synergistic inhibition by ATP and 2-oxoglutarate. As the same metabolites stimulate the P(II) kinase activity, it appears that the phosphorylation state of P(II) is determined by ATP and 2-oxoglutarate-dependent reciprocal reactivity of P(II) towards its phosphatase and kinase.

A NADP-glutamate dehydrogenase mutant of the petit-negative yeast Kluyveromyces lactis uses the glutamine synthetase-glutamate synthase pathway for glutamate biosynthesis.

The activities of the enzymes involved in ammonium assimilation and glutamate biosynthesis were determined in wild-type and NADP-glutamate dehydrogenase (GDH) null mutant strains of Kluyveromyces lactis. The specific NADP-GDH activity from K. lactis was fivefold lower than that found in Saccharomyces cerevisiae. The glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were similar to those reported in S. cerevisiae. The NADP-GDH null mutant was obtained by transforming the uraA strain MD2/1 with a linearized integrative yeast vector harbouring a 390 bp fragment of the NADP-GDH structural gene. This mutant grew as well as the parent strain on ammonium, but showed GS and GOGAT activities higher that those found in the wild-type strain, implying that the GS-GOGAT pathway could play a leading role in glutamate biosynthesis in K. lactis. Southern blotting analysis of K. lactis chromosomes separated by contour-clamped homogeneous electric field electrophoresis, indicated that the NADP-GDH structural gene is localized on chromosome VI.

Halotolerance of the Phototrophic Bacterium Rhodobacter capsulatus E1F1 Is Dependent on the Nitrogen Source

Phototrophic growth of the moderate halotolerant Rhodobacter capsulatus strain E1F1 in media containing up to 0.3 M NaCl was dependent on the nitrogen source used. In these media, increased growth rates and growth levels were observed in the presence of reduced nitrogen sources such as ammonium and amino acids. When the medium contained an oxidized nitrogen source (dinitrogen or nitrate), increases in salinity severely inhibited phototrophic growth. However, the addition of glycine betaine promoted halotolerance and allowed the cells to grow in 0.2 M NaCl. Inhibition of diazotrophic growth by salinity was due to a decrease in nitrogenase activity which was no longer synthesized and reversibly inactivated, both effects being alleviated by the addition of glycine betaine. In R. capsulatus E1F1, inhibition of cell growth in nitrate by salt was due to a rapid inhibition of nitrate uptake, which led to a long-term decrease in nitrate reductase activity, probably caused by repression of the enzyme. Addition of glycine betaine immediately restored nitrate uptake, but the recovery of nitrate reductase activity required several hours. Neither ammonium uptake nor ammonium assimilation through the glutamine synthetase-glutamate synthase pathway was affected by NaCl.

Effects of host feeding and dissolved ammonium on cell division and nitrogen status of zooxanthellae in the hydroid Myrionema amboinense

There is a relationship between host feeding, nitrogen status and mitotic activity of zooxanthellae symbiotic with the marine hydroid Myrionema amboinense. Decreases in the mitotic index of zooxanthellae in starved M. amboinense, and in internal pool sizes of glutamine and glutamate, amino acids involved in ammonium assimilation via the glutamine synthetase-glutamate synthase (GS/GOGAT) pathway, were partially restored by addition of ammonium chloride to seawater in which hydroids were incubated. Levels of glutamine were more sensitive to host starvation than levels of glutamate, resulting in a decrease in the glutamine: glutamate molar ratio to that found in zooxanthellae cultured on nitrate. Hydroids starved for 5 d and then incubated in different concentrations of ammonium chloride showed a positive correlation between ammonium concentration and mitotic index of their symbiotic zooxanthellae. Host starvation caused a decrease in perturbation of levels of glutamine and glutamate during ammonium assimilation, as well as decreases in rates of assimilation of [14C]-leucine into TCA-insoluble protein, and in photosynthetic incorporation of [14C]-bicarbonate. These observations suggest that host starvation reduces nitrogen supply to the zooxanthellae, causing nitrogen stress to the symbionts and reduction in metabolic processes associated with nitrogen assimilation and photosynthesis as well as with cell division.

Why does Escherichia coli have two primary pathways for synthesis of glutamate?

Escherichia coli has two primary pathways for glutamate synthetase-glutamate synthase pathway is known to be essential for synthesis at low ammonium concentrations and for regulation of the glutamine pool, but the necessity for glutamate dehydrogenase (GDH) has been uncertain. The results of competition experiments between the wild type and a GDH-deficient mutant during nutrient-limited growth and of direct enzyme measurements suggest that GDH is used in glutamate synthesis when the cell is limited for energy (and carbon) but ammonium and phosphate are present in excess, while the glutamine synthetase-glutamate synthase pathway is used when the cell is not under energy limitation. The use of alternative routes for glutamate synthesis implies that the energy cost of biosynthesis may be less when energy is limited than when energy is unlimited.

Assimilation of ethylenediamine as nitrogen source by the cyanobiont Nostoc ANTH

Nostoc ANTH metabolizes ethylenediamine (EDA) as sole nitrogen source but not as a carbon source. EDA is assimilated by the glutamine synthetase-glutamate synthase pathway. EDA represses heterocyst formation and nitrogenase activity but this is reversed by L-methionine-DL-sulphoximine.

Different ammonium-ion uptake, metabolism and detoxification efficiencies in two Lemnaceae

15N-Nuclear magnetic resonance spectroscopy was used to follow nitrogen assimilation and amino-acid production in Wolffia arrhiza (L.) Hork. ex. Wimmer, clone Golan exposed to 4.0 mM 15NH4Cl solutions for 24 h. The main 15N-labelled metabolites were asparagine and glutamine, as well as substantial amounts of unreacted, intracellular NH 4 + . These results were compared with those of a previous study on Lemna gibba L. clone Hurfeish (Monselise et al., 1987, New Phytol. 10, 341–345) with regard to NH 4 + uptake, assimilation and detoxification efficiencies. Both species, grown under continuous white light, were capable of preferential uptake of NH 4 + in the presence of nitrate. Relative growth rates indicate that both species tolerate increased levels of NH 4 + , up to 10−2 mol · 1−1, with L. gibba showing a slightly greater tolerance. No 15N-labelled free NH 4 + was detectable in L. gibba, while in W. arrhiza excess NH 4 + was found within the cells. This fact indicates that L. gibba is more efficient in detoxification than W. arrhiza, presumably because of inability of W. arrhiza to regenerate the “NH 4 + traps”, glutamate and aspartate, rapidly enough. This is also evident from the observation that addition of α-ketoglutarate to the medium caused nearly complete assimilation of intracellular NH 4 + in W. arrhiza. In both plants, addition of α-ketoglutarate increased both NH 4 + uptake and assimilation. Addition of l-methionine dl-sulfoximine, an inhibitor of glutamine synthetase inhibited NH 4 + assimilation, while addition of azaserine, an inhibitor of glutamate synthase, resulted in 15N incorporation into the glutamine-amide position only. These results are consistent with the glutamine synthetase-glutamate synthase pathway being the major route of NH 4 + assimilation in the two plants under the conditions used.

Nitrogen fixation by the thermophilic green sulfur bacterium Chlorobium tepidum.

The thermophilic green sulfur bacterium Chlorobium tepidum grew with N2, NH4+, or glutamine as the sole nitrogen source under phototrophic (anaerobic-light) conditions. Growth on N2 required increased buffering capacity to stabilize uncharacterized pH changes that occurred during diazotrophic growth. Increased sulfide levels were stimulatory for growth on N2. Levels of nitrogenase activity (acetylene reduction) in N2-grown C. tepidum cells were very high, among the highest ever reported for anoxygenic phototrophic bacteria. Maximal acetylene reduction rates in C. tepidum cells were observed at 48 to 50 degrees C, which is about 15 degrees C higher than the optimum temperature for nitrogenase activity in mesophilic chlorobia, and nitrogenase activity in C. tepidum responded to addition of ammonia by a "switch-off/switch-on" mechanism like that in phototrophic purple bacteria. C. tepidum cells assimilated ammonia mainly via the glutamine synthetase-glutamate synthase pathway, elevated levels of both of these enzymes being present in cells grown on N2. These results show that N2 fixation can occur in green sulfur bacteria up to at least 60 degrees C and that regulatory mechanisms important in control of nitrogenase activity in mesophilic anoxygenic phototrophs also appear to regulate thermally active forms of the enzyme.

Regulation of assimilatory nitrate reductase in the cyanobacteriumAnabaena doliolum

The assimilatory nitrate reductase (NR) from the cyanobacteriumAnabaena doliolum was membrane bound and solubilized by sonication. The Km value of the enzyme was 870 µM for nitrate with dithionite-reduced methyl viologen (MV) as electron donor. The pH optimum was 10.5 in the MV assay. Nitrate acted as an inducer and ammonium as repressor of the enzyme synthesis. In the presence ofl-methionine-d,l-sulfoximine (MSX) or azaserine, inhibitors of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway, ammonium did not exhibit any inhibitory effect on the enzyme. The photosynthetic nature of NR was shown with PS II inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). This enzyme fromA. doliolum has been shown to be a light-driven process, requiring de novo protein synthesis. It was inhibited by chlorate, the structural analog of nitrate;p-chloromercuribenzoate, a thiol reagent; sodium tungstate; and certain cations.

Ammonium assimilation in Proteus vulgaris, Bacillus pasteurii, and Sporosarcina ureae

No active uptake of ammonium was detected in Proteus vulgaris, Bacillus pasteurii, and Sporosarcina ureae, which indicates that these bacteria depend on the passive diffusion of ammonia across the cell membrane. In P. vulgaris the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway and glutamate dehydrogenase (GDH) were present, and these enzymes exhibited high affinities for ammonium. In B. pasteurii and S. ureae, however, no GS activity was detected, and GOGAT activity was only present in S. ureae. GDH enzymes were present in these two organisms, but showed only low affinity for ammonium, with apparent Km-values of 55.2 mM in B. pasteurii and 36.7 mM in S. ureae, respectively. These observations explain why P. vulgaris is able to grow at neutral pH and low ammonium concentration (2 mM), while B. pasteurii and S. ureae require high ammonium concentration (40 mM) and alkaline pH for growth.

Ammonium assimilation in Rhizobium phaseoli by the glutamine synthetase-glutamate synthase pathway.

Evidence from in vitro and in vivo studies showed that in Rhizobium phaseoli ammonium is assimilated by the glutamine synthetase (GS)-glutamate synthase NADPH pathway. No glutamate dehydrogenase activity was detected. R. phaseoli has two GS enzymes, as do other rhizobia. The two GS activities are regulated on the basis of the requirement for low (GSI) or high (GSII) ammonium assimilation. When the 2-oxoglutarate/glutamine ratio decreases, GSI is adenylylated. When GSI is inactivated, GSII is induced. However, induction of GSII activity varied depending on the rate of change of this ratio. GSII was inactivated after the addition of high ammonium concentrations, when the 2-oxoglutarate/glutamine ratio decreased rapidly. Ammonium inactivation resulted in alteration of the catalytic and physical properties of GSII. GSII inactivation was not relieved by shifting of the cultures to glutamate. After GSII inactivation, ammonium was excreted into the medium. Glutamate synthase activity was inhibited by some organic acids and repressed when cells were grown with glutamate as the nitrogen source.

Introduction of the Escherichia coli gdhA gene into Rhizobium phaseoli: effect on nitrogen fixation

Rhizobium phaseoli lacks glutamate dehydrogenase (GDH) and assimilates ammonium by the glutamine synthetase-glutamate synthase pathway. A strain of R. phaseoli harboring the Escherichia coli GDH structural gene (gdhA) was constructed. GDH activity was expressed in R. phaseoli in the free-living state and in symbiosis. Nodules with bacteroids that expressed GDH activity had severe impairment of nitrogen fixation. Also, R. phaseoli cells that lost GDH activity and assimilated ammonium by the glutamine synthetase-glutamate synthase pathway preferentially nodulated Phaseolus vulgaris.

GLUTAMINE SYNTHETASE AND AMMONIUM ASSIMILATION IN ROOTS OF ZINC‐TOLERANT AND NON‐TOLERANT CLONES OF DESCHAMPSIA CESPITOSA (L.) BEAUV. AND ANTHOXANTHUM ODORATUM L.

SummaryThe influence of zinc on glutamine synthetase (GS) activity and ammonium assimilation in roots of zinc‐tolerant and non‐tolerant clones of Deschampsia cespitosa (L.) Beauv. and Anthoxanthum odoratum L. was studied. One isoform of GS was resolved by ion exchange chromatography in D. cespitosa roots. However, two isoforms were found in A. odoratum roots; one of these was attributed to fungal contamination and was not studied further. There was no difference in the sensitivity of partially purified GS from zinc‐tolerant and non‐tolerant clones to inhibition by zinc. Inhibition of GS by Zn was reversible and the kinetics of inhibition were studied. Methionine sulphoximine (an inhibitor of GS) induced ammonium accumulation in the roots of control and zinc‐grown roots of Zn‐tolerant D. cespitosa. Zinc inhibited incorporation of [15N]ammonium into free amino acids (determined by combined gas chromatography–mass spectrometry) in both clones of D. cespitosa, but higher concentrations were required to achieve inhibition in the tolerant clone. The labelling pattern was unaffected by zinc in both clones. The results suggest that zinc has no effect on the pathway of ammonium assimilation in zinc‐grown roots and that assimilation is via the glutamine synthetase–glutamate synthase pathway. There was no evidence to support the hypothesis that glutamate dehydrogenase functions preferentially in ammonium assimilation under conditions of environmental stress.

Utilization of Ammonium by Mutant and Wild Types of Chlamydomonas reinhardii. Studies of the Glutamate Synthase Activities

The utilization of ammonium by Chlamydomonas is mainly a light-dependent process, mediated by the glutamine synthetase-glutamate synthase pathway. Ferredoxin-dependent and NADH-glutamate synthase activities were measured in Chlamydomonas cells treated with toluene. The assay with reduced methyl viologen as electron donor measures the sum of the two activities. The Fd- and NADH-glutamate synthase activities respond differently to several nutritional conditions consistent with different physiological roles for the two enzymes. The Fd-glutamate synthase is probably responsible for the photosynthetic assimilation of ammonium and the reassimilation of ammonium formed during photorespiration. On the other hand, the NADH-glutamate synthase may be involved more in the assimilation of ammonium in the dark and in the recycling of ammonium released by protein metabolism under low availability of nitrogen.