Robustness in Escherichia coli Glutamate and Glutamine Synthesis Studied by a Kinetic Model

Metabolic control of glutamine and glutamate synthesis from ammonia and oxoglutarate in Escherichia coli is tight and complex. In this work, the role of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) regulation in this control was studied. Both enzymes form a linear pathway, which can also have a cyclic topology if glutamate–oxoglutarate amino transferase (GOGAT) activity is included. We modelled the metabolic pathways in the linear or cyclic topologies using a coupled nonlinear differential equations system. To simulate GS regulation by covalent modification, we introduced a relationship that took into account the levels of oxoglutarate and glutamine as signal inputs, as well as the ultrasensitive response of enzyme adenylylation. Thus, by including this relationship or not, we were able to model the system with or without GS regulation. In addition, GS and GDH activities were changed manually. The response of the model in different stationary states, or under the influence of N-input exhaustion or oscillation, was analyzed in both pathway topologies. Our results indicate a metabolic control coefficient for GDH ranging from 0.94 in the linear pathway with GS regulation to 0.24 in the cyclic pathway without regulation, employing a default GDH concentration of 8 μM. Thus, in these conditions, GDH seemed to have a high degree of control in the linear pathway while having limited influence in the cyclic one. When GS was regulated, system responses to N-input perturbations were more sensitive, especially in the cyclic pathway. Furthermore, we found that effects of regulation against perturbations depended on the relative values of the glutamine and glutamate output first-order kinetic constants, which we named k ₆ and k ₇, respectively. Effects of regulation grew exponentially with a factor around 2, with linear increases of (k ₇???k ₆). These trends were sustained but with lower differences at higher GS concentration. Hence, GS regulation seemed important for metabolic stability in a changing environment, depending on the cell’s metabolic status.     

Arginase, glutamine synthetase and glutamate dehydrogenase activities in moist chilled and warm-incubated walnut kernels

The activities of arginase, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were studied in both moist chilled (5°C) and warm (27°C) incubated walnut (Juglans regia. L) kernels to asses whether the non-germinability of dormant kernels is associated with failure in amino acid metabolism. Warm-incubated kernels showed low germination (25%), whereas cold-stratified kernels displayed germination up to 61%. Arginase activity increased about twofold in imbibed kernels. It remained at a high level in cold-stratified kernels from mid-period of incubation onwards; however, in warm-incubated kernels the activity declined after an initial increase so that by 20 days, it was negligible. No significant differences in GS activity occurred between cold-stratified and warm-incubated kernels, but the activity of GDH was significantly more in kernels incubated at warm conditions. Thin-layer chromatographic separation of polyamines revealed greater ammonia, spermidine and an unknown polyamine accumulation in warm-incubated kernels. Thus, the declined rate of walnut kernel germination under warm conditions is mainly correlated with rapid inactivation of arginase, greater levels of ammonia and alterations in kernel polyamine composition. The enhanced activity of GDH in warm-incubated kernels implies that catabolic deamination of amino acids and their subsequent respiration is the favored pathway ongoing under warm conditions. This situation compromises germination-specific metabolism of amino acids which likely to operate better at lower temperatures during cold stratification of kernels.     

Molecular analyses of tomato GS,GOGAT and GDH gene families and their response to abiotic stresses

Glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) are closely related enzymes in plant nitrogen metabolism and potential targets for improving nitrogen use efficiency. However, little research has focused on the enzyme-encoding genes in tomato. Here, a comprehensive study of these genes was conducted. Six GS genes, two GOGAT genes and five GDH genes were identified in tomato. Bioinformatics and gene expression analyses suggested that these genes evolved species-specific regulatory properties and biological functions in tomato. SlNADH-GOGAT, SlGS1.1 and SlNAD-GDHB1 were abundantly expressed in roots, SlGS1.1 can be induced by nitrogen deprivation, and SlGS1.2, SlGS1.3, SlNADH-GOGAT and SlNAD-GDHB1 can be induced by the re-supply of nitrogen after 5 days of deprivation, they may play key roles in primary nitrogen assimilation. SlFd-GOGAT, SlGS1.1 and SlNAD-GDHA1-A2 were also highly expressed in fruits, indicating their important roles in fruit development and ripening. Tomato GS, GOGAT and GDH may be involved in stress responsiveness, since most of these genes modified their expression levels under drought, cold or heat stress treatment. We believe these findings will assist in the exploration of the genes’ biological functions and regulatory mechanisms, as well as the studies to improve nitrogen use efficiency, stress resistance and fruit quality in tomato.     

氮素水平对花生氮素代谢及相关酶活性的影响

 在大田高产条件下研究了氮素水平对花生(Arachis hypogaea)可溶性蛋白质、游离氨基酸含量及氮代谢相关酶活性的影响, 结果表明, 适当提高氮素水平既能增加花生各器官中可溶性蛋白质和游离氨基酸的含量, 又能提高硝酸还原酶、谷氨酰胺合成酶和谷氨酸脱氢酶等氮素同化酶的活性, 使其达到同步增加; 氮素水平过高虽能提高硝酸还原酶和籽仁蛋白质含量, 但谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH)的活性下降; N素施肥水平不改变花生植株各器官中可溶性蛋白质、游离氨基酸含量以及硝酸还原酶(NR)、谷氨酰胺合成酶、谷氨酸脱氢酶活性的变化趋势, 但适量施N (A2和A3处理)使花生各营养器官中GS、GDH活性提高; 氮素水平对花生各叶片和籽仁中GS、GDH活性的高低影响较大, 但对茎和根中GDH活性大小的影响较小。     

Over-expression of a fungal NADP(H)-dependent glutamate dehydrogenase PcGDH improves nitrogen assimilation and growth quality in rice

Glutamate dehydrogenase (GDH) tends to have a lower affinity for ammonium than glutamine synthetase (GS) in higher plants. Consequently, nitrogen is mostly assimilated as ammonium by the GS/glutamate synthase pathway which requires 2-oxoglutarate (2-OG) as carbon skeletons. In contrast, the NADP(H)-dependent GDH in fungi has a higher affinity for ammonium than that in higher plants and plays a more significant part in ammonium assimilation. We isolated an NADP(H)-GDH gene (PcGDH) from the fungus Pleurotus cystidiosus and heterologously expressed it in rice (Oryza sativa L.). Alterations in nitrogen assimilation, growth, metabolism, and grain yield were observed in the transgenic plants. An investigation of the kinetic properties of the purified recombinant protein demonstrated that the amination activity (7.05 ± 0.78 μmoL min^?1 mg soluble protein^?1) of PcGDH was higher than the deamination activity (3.36 ± 0.42 μmoL min^?1 mg soluble protein^?1) and that the K _m value for ammonium (K _m = 3.73 ± 0.23 mM) was lower than that for the glutamate (K _m = 15.97 ± 0.31 mM), indicating that the PcGDH tends to interconvert 2-OG and glutamate. Examination of the activity of NADP(H)-GDH in control and transgenic lines demonstrated that NADP(H)-GDH activity in the transgenic lines was markedly higher than that in the control lines; in particular, the amination activity was significantly higher than the deamination activity in shoots of the transgenic lines. The results of the hydroponics experiment revealed that shoot and root length, fresh weight, chlorophyll content, nitrogen content, and amino acid levels (glutamate, glutamine, and total amino acids) were elevated in transgenic lines in comparison with those of the control line under different nitrogen conditions at seedling stage. The 1,000-grain weight and the panicle number in transgenic lines were considerably augmented in the field condition, yet the filled grain rate dropped slightly and there was no apparent change in the grain yield. The levels of glutelin and prolamine in the transgenic seeds were considerably higher than those in control seeds. In conclusion, these results demonstrate that heterologous expression of P. cystidiosus GDH (PcGDH) could improve nitrogen assimilation and growth in rice.     

Genetic and physiological analysis of germination efficiency in maize in relation to nitrogen metabolism reveals the importance of cytosolic glutamine synthetase

We have developed an approach combining physiology and quantitative genetics to enhance our understanding of nitrogen (N) metabolism during kernel germination. The physiological study highlighted the central role of glutamine (Gln) synthetase (GS) and Gln synthesis during this developmental process because a concomitant increase of both the enzyme activity and the amino acid content was observed. This result suggests that Gln is acting either as a sink for ammonium released during both storage protein degradation and amino acid deamination or as a source for amino acid de novo synthesis by transamination. In the two parental lines used for the quantitative genetics approach, we found that the increase in Gln occurred earlier in Io compared with F(2), a result consistent with its faster germinating capacity. The genetic study was carried out on 140 F6 recombinant inbred lines derived from the cross between F(2) and Io. Quantitative trait locus mapping identified three quantitative trait loci (QTLs) related to germination trait (T50, time at which 50% of the kernels germinated) that explain 18.2% of the phenotypic variance; three QTLs related to a trait linked to germination performance, kernel size/weight (thousand kernels weight), that explain 17% of the phenotypic variance; two QTLs related to GS activity at early stages of germination that explain 17.7% of the phenotypic variance; and one QTL related to GS activity at late stages of germination that explains 7.3% of the phenotypic variance. Coincidences of QTL for germination efficiency and its components with genes encoding cytosolic GS (GS1) and the corresponding enzyme activity were detected, confirming the important role of the enzyme during the germination process. A triple colocalization on chromosome 4 between gln3 (a structural gene encoding GS1) and a QTL for GS activity and T50 was found; whereas on chromosome 5, a QTL for GS activity and thousand kernels weight colocalized with gln4, another structural gene encoding GS1. This observation suggests that for each gene, the corresponding enzyme activity is of major importance for germination efficiency either through the size of the grain or through its faster germinating capacity. Consistent with the possible nonoverlapping function of the two GS1 genes, we found that in the parental line Io, the expression of Gln3 was transiently enhanced during the first hours of germination, whereas that of gln4 was constitutive.     

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     

The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicotiana tabacum L. leaves

To investigate the role of stress in nitrogen management in plants, the effect of pathogen attack, elicitors, and phytohormone application on the expression of the two senescence-related markers GS1 (cytosolic glutamine synthetase EC 6.3.1.2) and GDH (glutamate dehydrogenase, EC 1.4.1.2) involved in nitrogen mobilization in senescing leaves of tobacco (Nicotiana tabacum L.) plants, was studied. The expression of genes involved in primary nitrogen assimilation such as GS2 (chloroplastic glutamine synthetase) and Nia (nitrate reductase, EC 1.6.1.1) was also analysed. The Glubas gene, coding a beta-1,3-glucanase, was used as a plant-defence gene control. As during natural senescence, the expression of GS2 and Nia was repressed under almost all stress conditions. By contrast, GS1 and GDH mRNA accumulation was increased. However, GS1 and GDH showed differential patterns of expression depending on the stress applied. The expression of GS1 appeared more selective than GDH. Results indicate that the GDH and GS1 genes involved in leaf senescence are also a component of the plant defence response during plant-pathogen interaction. The links between natural plant senescence and stress-induced senescence are discussed, as well as the potential role of GS1 and GDH in a metabolic safeguard process.     

The pivotal role of glutamate dehydrogenase (GDH) in the mobilization of N and C from storage material to asparagine in germinating seeds of yellow lupine

In germinating seeds of legumes, amino acids liberated during mobilization of storage proteins are partially used for synthesis of storage proteins of the developing axis, but some of them are respired. The amino acids are catabolized by both glutamate dehydrogenase (GDH) and transaminases. Ammonium is reassimilated by glutamine synthetase (GS) and, through the action of asparagine synthetase (AS), is stored in asparagine (Asn).This review presents the ways in which amino acids are converted into Asn and their regulation, mostly in germinating seeds of yellow lupine, where Asn can make up to 30% of dry matter. The energy balance of the synthesis of Asn from glutamate, the most common amino acid in lupine storage proteins, also shows an adaptation of lupine for oxidation of amino acids in early stages of germination.Regulation of the pathway of Asn synthesis is described with regard to the role of GDH and AS, as well as compartmentation of particular metabolites. The regulatory effect of sugar on major links of the pathway (mobilization of storage proteins, induction of genes and activity of GDH and AS) is discussed with respect to recent genetic and molecular studies. Moreover, the effect of glutamate and phytohormones is presented at various stages of Asn biosynthesis.     

Modulation of key nitrogen assimilating enzymes by NAA and in vitro culture in Cuscuta reflexa

Nitrogen assimilation in the callus of an angiosperm holoparasitic plant, Cuscuta reflexa, has been investigated by studying the level of key enzymes of the nitrogen assimilation pathway, namely nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH), during its growth in the absence and presence of NAA. The activity of all these enzymes in culture exhibited a developmental profile of an initial increase followed by a decrease. The presence of NAA increased the activity of all the enzymes throughout the culture period without altering their developmental profiles. Isozyme profiles of GS and GDH in the callus of C. reflexa were analyzed by PAGE and direct in gel activity staining. In the absence of NAA, the callus exhibited one isozyme of GS and two isozymes of GDH. NAA treatment led to the development of one additional isozyme of GS. Further stimulating effect of NAA on the activity of each of these enzymes was also evident by in gel activity staining of the isozymes. A comparison of the levels of NR, GS, GOGAT and GDH in field vines of C. reflexa, leaves of its host plant, Catheranthus with those of Cuscuta callus, led to the observation that all the nitrogen assimilating enzymes except GDH, were absent in the field vines of C. reflexa. Callus and field vines revealed a preponderance of GDH as compared to GS activity, while a reverse trend was observed in the host plant. The data are suggestive of ammonia assimilation through GDH pathway in this parasite.     

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.     

Glutamate dehydrogenase and glutamine synthetase activities during the development of triticale grains

Glutamate dehydrogenase (GDH, EC 1.4.1.2–4) and glutamine synthetase (GS, EC 6.3.1.2) activities as well as protein content and dry matter in developing kernels of winter Triticale were determined. The relatively low level of GS activity compared to high level of NAD(P)H-dependent GDH activity during intensive filling of grains with storage compounds may indicate the participation of GDH in reductive amination of 2-oxoglutarate. The amination activity of this enzyme in all grain development phases exceeded the deaminating activity several fold. Moreover, the dynamics in the change of NAD(P)H-GDH and NAD(P)⁺-GDH activities were analysed in various tissues of the developing grains. The high amination activity of the enzyme in the seed coat, where the intensive protein synthesis occurs would also be an indication of the anabolic function of this enzyme.     

Glutamine synthetase and glutamate dehydrogenase in cadmium-stressed triticale seedlings

The studies were performed on young triticale seedlings grown on a mineral medium containing 5 mM NO ₃ ⁻ as the nitrogen source, with the addition of 0.5 mM CdCl₂. It was determined that cadmium ions accumulated mainly in the plant roots. Decreases in nitrate concentrations both in the roots and shoots of seedlings, as well as decreases in soluble protein contents with simultaneous increases in endopeptidase activity were also observed. Both in roots and shoots significant decreases in glutamic acid were noted. Toxic cadmium ion accumulation in seedlings significantly modified activity of primary nitrogen assimilating enzymes, i.e. glutamine synthetase (GS, EC 6.3.1.2) and glutamate dehydrogenase (GDH, EC 1.4.1.2). There was a significant decrease in GS activity both in roots and in shoots of the stressed plants, in comparison to plants grown without cadmium. In shoots of the control plants and plants subjected to stress two GS isoforms were discovered: cytoplasmatic (GS₁) and chloroplastic (GS₂). Substantial decreases in total glutamine synthetase activity in green parts of seedlings, occurring under stress conditions, result from dramatic decrease in GS₂ activity (by 60 % in relation to the control plants); despite simultaneous increases in the cytoplasmatic isoform (GS₁) activity by approx. 96 %. Cadmium ions accumulating in roots and shoots of seedlings not only increased GDH activity, but also modified its coenzymatic specificity.     

Root and Nodule Enzymes of Ammonia Assimilation in Two Plant-Conditioned Symbiotically Ineffective Genotypes of Alfalfa (Medicago sativa L.)

Biochemical and physiological parameters associated with nitrogen metabolism were measured in nodules and roots of glasshouse-grown clones of two symbiotically ineffective alfalfa (Medicago sativa L.) genotypes supplied with either NO₃⁻ or NH₄⁺. Significant differences were observed between genotypes for nodule soluble protein concentrations and glutamine synthetase (GS) and glutamate synthase (GOGAT) specific activities, both in untreated controls and in response to applied N. Nodule soluble protein of both genotypes declined in response to applied N, while nodule GS, GOGAT, and glutamate dehydrogenase (GDH) specific activities either decreased or remained relatively constant. In contrast, no genotype differences were observed in roots for soluble protein concentrations and GS, GOGAT, and GDH specific activities, either in untreated controls or in response to applied N. Root soluble protein levels and GS and GOGAT specific activities of N-treated plants increased 2- to 4-fold within 4 days and then decreased between days 13 and 24. Root GDH specific activity of NH₄⁺-treated plants increased steadily throughout the experiment and was 50 times greater than root GS or GOGAT specific activities by day 24.     

Glutamate dehydrogenase of the germinating triticale seeds: gene expression,activity distribution and kinetic characteristics

On the cross-roads of main carbon and nitrogen metabolic pathways, glutamate dehydrogenase (GDH, E.C. 1.4.1.2) carries out the reaction of reductive amination of 2-oxoglutarate to glutamate (the anabolic activity; NAD(P)H–GDH), and the reverse reaction of oxidative deamination of glutamic acid (the catabolic activity; NAD(P)⁺–GDH). To date, there have been no reports on identification of GDH genes in cereals. Here, we report cloning and biochemical characterization of the GDH from germinating triticale seeds, a common Polish cereal. A single TsGDH1 gene is 1,620 bp long, while its 1,236 bp long open reading frame encodes a protein of 411 amino acids of high homology with the published GDH protein sequences from other plants. Phylogenetic analyses locate the TsGDH1 among other monocotyledonous proteins and among the sequences of the β-type subunit of plant GDHs. Changes in TsGDH1 expression and the dynamics of enzyme activity in germinating seeds confirm the existence of one TsGDH isoform with varying expression and activity patterns, depending on the tissue localization and stage of germination. The four-step purification method (including the anionite chromatography using HPLC) resulted in a protein preparation with a high-specific activity and purification factor of approx. 230. The purified enzyme exhibited an absolute specificity towards 2-oxoglutarate (NAD(P)H–GDH), or towards l-glutamate in the reverse reaction (NAD(P)⁺–GDH), while its low K _m constants towards all substrates and co-enzymes may suggest its aminating activity during germination, or, alternatively, its capability to adjust the direction of the catalyzed reaction according to the metabolic necessity.     

Evaluation of the enzymes involved in primary nitrogen metabolism in Tilia platyphyllos-Tuber borchii ectomycorrhizae

No information is available about Tuber borchii Vittad. ammonium metabolism during its life cycle, which involves the succession of three distinct phases. In this direction, the levels of glutamine synthetase (GS; EC 6.3.1.2), glutamate synthase (GOGAT; EC 1.4.1.13-14) and glutamate dehydrogenase (GDH; EC 1.4.1.2-4) were evaluated in Tilia platyphyllos Scop.-Tuber borchii Vittad. ectomycorrhizae, free living mycelium and non-inoculated roots. In the plant roots, GS shows high specific activity and only NADH-GDH (EC 1.4.1.2) is detectable; on the other hand, in free living mycelium GS and NADPH-GDH (EC 1.4.1.4) can be detected. Ectomycorrhizal metabolism was found to be deeply influenced by the two symbiotic partners. In fact, GS and both forms of GDH are present and their specific activities are higher than those found in the plant root and in the mycelial cells.