The crystal structure of phosphinothricin in the active site of glutamine synthetase illuminates the mechanism of enzymatic inhibition

Phosphinothricin is a potent inhibitor of the enzyme glutamine synthetase (GS). The resolution of the native structure of GS from Salmonella typhimurium has been extended to 2.5 A resolution, and the improved model is used to determine the structure of phosphinothricin complexed to GS by difference Fourier methods. The structure suggests a noncovalent, dead-end mechanism of inhibition. Phosphinothricin occupies the glutamate substrate pocket and stabilizes the Glu327 flap in a position which blocks the glutamate entrance to the active site, trapping the inhibitor on the enzyme. One oxygen of the phosphinyl group of phosphinothricin appears to be protonated, because of its proximity to the carboxylate group of Glu327. The other phosphinyl oxygen protrudes into the negatively charged binding pocket for the substrate ammonium, disrupting that pocket. The distribution of charges in the glutamate binding pocket is complementary to those of phosphinothricin. The presence of a second ammonium binding site within the active site is confirmed by its analogue thallous ion, marking the ammonium site and its protein ligands. The inhibition of GS by methionine sulfoximine can be explained by the same mechanism. These models of inhibited GS further illuminate its catalytic mechanism.     

Computational Structural Analysis and Kinetic Studies of a Cytosolic Glutamine Synthetase from Camellia sinensis (L.) O. Kuntze

Structural analysis of a cytosolic glutamine synthetase from Camellia sinensis (CsGS) has been conducted employing computational techniques. This was conducted to compare its structural aspects with other known structures of GS. The disordered residues and their distribution in CsGS are in close comparison to earlier reported GS. The 3-D structure of CsGS also showed high degree of similarity with the only known crystal structure of GS from Zea mays. The K _m values observed with recombinant CsGS for all the three substrates are higher compared to rice, Arabidopsis, maize and human. This suggests lower affinity of CsGS for substrates. Further, kinetic mechanism of CsGS catalysis was investigated using initial velocity analysis and product inhibition studies. Initial velocity data eliminate the possibility of ping-pong mechanism and favor the random mechanism of catalysis. Through product inhibition studies, ADP was found to be a competitive inhibitor with respect to ATP and noncompetitive inhibitor versus both glutamate and ammonium. While, glutamine and inorganic phosphate were found to be non-competitive inhibitors of ATP, glutamate and ammonia. Taken together, these observations are consistent with a random catalysis mechanism for the CsGS where the binding order of certain substrates is kinetically preferred by the enzyme.     

Oxidative modification of Escherichia coli glutamine synthetase. Decreases in the thermodynamic stability of protein structure and specific changes in the active site conformation.

Metal catalyzed oxidation of specific amino acid residues has been proposed to be an important physiological mechanism of marking proteins for proteolytic degradation. After initial oxidative inactivation of dodecameric Escherichia coli glutamine synthetase (GS), the integrity of the GS active site and protein structure was assessed by monitoring ATP binding, observing a susceptibility of GS to tryptic cleavage, and comparative thermodynamic analysis. The tryptic cleavage rates of an active site linked central loop were significantly accelerated for the oxidized conformer. This tryptic cleavage was essentially prevented in the presence of glutamate for native GS but not for the oxidized conformer. The integrity of the ATP binding site in the oxidized GS was substantially altered as indicated by the reduction in fluorescence enhancement associated with ATP binding. Decreases in the free energies of quaternary protein structure and subunit interactions due to oxidative modification were determined by temperature and urea induced unfolding equilibrium measurements. Comparative thermal stability measurements of a partial unfolding transition indicated that the loss in stabilization free energy for the oxidized GS conformer was 1.3 kcal/mol dodecamer. Under alkaline conditions, the urea-induced disruption of quaternary and tertiary structures of oxidized and native GS were examined. This comparative analysis revealed that the free energies of the subunit interactions and unfolding of the dissociated monomers for oxidized GS were decreased by 1.5 and 1.7 kcal/mol, respectively. Our results suggest that small free energy decreases in GS protein structural stability of only 1-2 kcal/mol may be responsible for the selective proteolytic turnover of the oxidized GS.     

Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design

Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 Å. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 Å and 2.6 Å, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.     

Hairy roots of Brassica napus: I. Applied glutamine overcomes the effect of phosphinothricin treatment

Summary Hairy roots of Brassica napus (rape cv. Giant) were produced by cocultivating leaf and cotyledon explants with Agrobacterium rhizogenes strain A4T. The hairy roots grew prolifically on solid and in liquid media. Incorporation of ammonium sulphate or phosphinothricin (PPT) into the media reduced growth. PPT treatment reduced glutamine synthetase (GS) activity and increased the ammonia content of the hairy roots. We have found that PPT treatment also induces a loss of glutamine from the roots and this may influence root growth. To test this we grew hairy roots in a liquid medium containing 10 mM glutamine. This glutamine treatment overcame the PPT induced suppression of growth but also significantly increased GS activity, reduced ammonia accumulation and increased the levels of glutamate and asparagine.     

YbdK is a carboxylate-amine ligase with a gamma-glutamyl:Cysteine ligase activity: crystal structure and enzymatic assays

The Escherichia coli open reading frame YbdK encodes a member of a large bacterial protein family of unknown biological function. The sequences within this family are remotely related to the sequence of gamma-glutamate-cysteine ligase (gamma-GCS), an enzyme in the glutathione biosynthetic pathway. A gene encoding gamma-GCS in E. coli is already known. The 2.15 A resolution crystal structure of YbdK reveals an overall fold similar to that of glutamine synthetase (GS), a nitrogen metabolism enzyme that ligates glutamate and ammonia to yield glutamine. GS and gamma-GCS perform related chemical reactions and require ATP and Mg2+ for their activity. The Mg2+-dependent binding of ATP to YbdK was confirmed by fluorescence spectroscopy employing 2'(or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate, and yielding a dissociation constant of 3 +/- 0.5 microM. The structure of YbdK contains a crevice that corresponds to the binding sites of ATP, Mg2+ and glutamate in GS. Many of the GS residues that coordinate the metal ions and interact with glutamic acid and the phosphoryl and ribosyl groups of ATP are also present in YbdK. GS amino acids that have been associated with ammonia binding have no obvious counterparts in YbdK, consistent with a substrate specificity that is different from that of GS. Ligase activity between glutamic acid and each of the twenty amino acid residues was tested on high performance liquid chromatography (HPLC) by following the hydrolysis of ATP to ADP. Catalysis was observed only with cysteine. A pyruvate kinase/lactic acid dehydrogenase coupled assay was used to rule out GS activity and to determine that YbdK exhibits gamma-GCS activity. The catalytic rate was found to be approximately 500-fold slower than that reported for authentic gamma-GCS.     

Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies

Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.     

Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site.

Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia and glutamate to yield glutamine, ADP, and inorganic phosphate in the presence of divalent cations. Bacterial GS is an enzyme of 12 identical subunits, arranged in two rings of 6, with the active site between each pair of subunits in a ring. In earlier work, we have reported the locations within the funnel-shaped active site of the substrates glutamate and ATP and of the two divalent cations, but the site for ammonia (or ammonium) has remained elusive. Here we report the discovery by X-ray crystallography of a binding site on GS for monovalent cations, Tl+ and Cs+, which is probably the binding site for the substrate ammonium ion. Fourier difference maps show the following. (1) Tl+ and Cs+ bind at essentially the same site, with ligands being Glu 212, Tyr 179, Asp 50', Ser 53' of the adjacent subunit, and the substrate glutamate. From its position adjacent to the substrate glutamate and the cofactor ADP, we propose that this monovalent cation site is the substrate ammonium ion binding site. This proposal is supported by enzyme kinetics. Our kinetic measurements show that Tl+, Cs+, and NH4+ are competitive inhibitors to NH2OH in the gamma-glutamyl transfer reaction. (2) GS is a trimetallic enzyme containing two divalent cation sites (n1, n2) and one monovalent cation site per subunit. These three closely spaced ions are all at the active site: the distance between n1 and n2 is 6 A, between n1 and Tl+ is 4 A, and between n2 and Tl+ is 7 A. Glu 212 and the substrate glutamate are bridging ligands for the n1 ion and Tl+. (3) The presence of a monovalent cation in this site may enhance the structural stability of GS, because of its effect of balancing the negative charges of the substrate glutamate and its ligands and because of strengthening the "side-to-side" intersubunit interaction through the cation-protein bonding. (4) The presence of the cofactor ADP increases the Tl+ binding to GS because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution.     

The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops

This short review outlines the central role of glutamine synthetase (GS) in plant nitrogen metabolism and discusses some possibilities for crop improvement. GS functions as the major assimilatory enzyme for ammonia produced from N fixation, and nitrate or ammonia nutrition. It also reassimilates ammonia released as a result of photorespiration and the breakdown of proteins and nitrogen transport compounds. GS is distributed in different subcellular locations (chloroplast and cytoplasm) and in different tissues and organs. This distribution probably changes as a function of the development of the tissue, for example, GS1 appears to play a key role in leaf senescence. The enzyme is the product of multiple genes with complex promoters that ensure the expression of the genes in an organ- and tissue-specific manner and in response to a number of environmental variables affecting the nutritional status of the cell. GS activity is also regulated post-translationally in a manner that involves 14-3-3 proteins and phosphorylation. GS and plant nitrogen metabolism is best viewed as a complex matrix continually changing during the development cycle of plants. Along with GS, a number of other enzymes play key roles in maintaining the balance of carbon and nitrogen. It is proposed that one of these is glutamate dehydrogenase (GDH). There is considerable evidence for a GDH shunt to return the carbon in amino acids back into reactions of carbon metabolism and the tri-carboxylic acid cycle. Results with transgenic plants containing transferred GS genes suggest that there may be ways in which it is possible to improve the efficiency with which crop plants use nitrogen. Marker-assisted breeding may also bring about such improvements.     

Glutamine synthetase in brain: effect of ammonia

Glutamine synthetase (GS) in brain is located mainly in astrocytes. One of the primary roles of astrocytes is to protect neurons against excitotoxicity by taking up excess ammonia and glutamate and converting it into glutamine via the enzyme GS. Changes in GS expression may reflect changes in astroglial function, which can affect neuronal functions.Hyperammonemia is an important factor responsible of hepatic encephalopathy (HE) and causes astroglial swelling. Hyperammonemia can be experimentally induced and an adaptive astroglial response to high levels of ammonia and glutamate seems to occur in long-term studies. In hyperammonemic states, astroglial cells can experience morphological changes that may alter different astrocyte functions, such as protein synthesis or neurotransmitters uptake. One of the observed changes is the increase in the GS expression in astrocytes located in glutamatergic areas. The induction of GS expression in these specific areas would balance the increased ammonia and glutamate uptake and protect against neuronal degeneration, whereas, decrease of GS expression in non-glutamatergic areas could disrupt the neuron-glial metabolic interactions as a consequence of hyperammonemia.Induction of GS has been described in astrocytes in response to the action of glutamate on active glutamate receptors. The over-stimulation of glutamate receptors may also favour nitric oxide (NO) formation by activation of NO synthase (NOS), and NO has been implicated in the pathogenesis of several CNS diseases. Hyperammonemia could induce the formation of inducible NOS in astroglial cells, with the consequent NO formation, deactivation of GS and dawn-regulation of glutamate uptake. However, in glutamatergic areas, the distribution of both glial glutamate receptors and glial glutamate transporters parallels the GS location, suggesting a functional coupling between glutamate uptake and degradation by glutamate transporters and GS to attenuate brain injury in these areas.In hyperammonemia, the astroglial cells located in proximity to blood-vessels in glutamatergic areas show increased GS protein content in their perivascular processes. Since ammonia freely crosses the blood-brain barrier (BBB) and astrocytes are responsible for maintaining the BBB, the presence of GS in the perivascular processes could produce a rapid glutamine synthesis to be released into blood. It could, therefore, prevent the entry of high amounts of ammonia from circulation to attenuate neurotoxicity. The changes in the distribution of this critical enzyme suggests that the glutamate-glutamine cycle may be differentially impaired in hyperammonemic states.     

Genome organization of glutamine synthetase genes in rainbow trout (Oncorhynchus mykiss)

Unlike mammals, bony fish appear to possess multiple genes encoding glutamine synthetase (GS), the nitrogen metabolism enzyme responsible for the conversion of glutamate and ammonia into glutamine at the expense of ATP. This study reports on the development of genetic markers for each of the four isoforms identified thus far in rainbow trout (Oncorhynchus mykiss) and their genome localization by linkage mapping. We found that genes coding for GS01, GS02, GS03, and GS04 map to four different linkage groups in the trout genome, namely RT-24, RT-23, RT-08, and RT-13, respectively. Linkage groups RT-23 and RT-13 appear to represent distinct chromosomes sharing duplicated marker regions, which lends further support to the previous suggestion that GS02 and GS04 may be duplicate gene copies that evolved from a whole-genome duplication in the trout ancestor. In contrast, there is at present no further evidence that RT-24 and RT-08 share ancestrally homologous segments and additional genomic studies will be needed to clarify the evolutionary origin of genes coding for GS01 and GS03.     

Glutamine synthetase activity in the ruminal bacterium Succinivibrio dextrinosolvens.

Succinivibrio dextrinosolvens C18 was found to possess glutamine synthetase (GS), urease, glutamate dehydrogenase, and several other nitrogen assimilation enzymes. When grown in continuous culture under ammonia limitation, both GS and urease activities were high and glutamate dehydrogenase activity was low, but the opposite activity pattern was observed for growth in the presence of ample ammonia. The addition of high-level (15 mM) ammonium chloride to ammonia-limited cultures resulted in a rapid loss of GS activity as measured by either the gamma-glutamyl transferase or forward assay method with cells or extracts. No similar activity losses occurred for urease, glutamate dehydrogenase, or pyruvate kinase. The GS activity loss was not prevented by the addition of chloramphenicol and rifampin. The GS activity could be recovered by washing or incubating cells in buffer or by the addition of snake venom phosphodiesterase to cell extracts. Manganese inhibited the GS activity (forward assay) of untreated cells but stimulated the GS activity in ammonia-treated cells. Alanine, glycine, and possibly serine were inhibitory to GS activity. Optimal pH values for GS activity were 7.3 and 7.4 for the forward and gamma-glutamyl transferase assays, respectively. The glutamate dehydrogenase activity was NADPH linked and optimal in the presence of KCl. The data are consistent with an adenylylation-deadenylylation control mechanism for GS activity in S. dextrinosolvens, and the GS pathway is a major route for ammonia assimilation under low environmental ammonia levels. The rapid regulation of the ATP-requiring GS activity may be of ecological importance to this strictly anaerobic ruminal bacterium.     

Glutamine synthetase activity in the ruminal bacterium Succinivibrio dextrinosolvens

Succinivibrio dextrinosolvens C18 was found to possess glutamine synthetase (GS), urease, glutamate dehydrogenase, and several other nitrogen assimilation enzymes. When grown in continuous culture under ammonia limitation, both GS and urease activities were high and glutamate dehydrogenase activity was low, but the opposite activity pattern was observed for growth in the presence of ample ammonia. The addition of high-level (15 mM) ammonium chloride to ammonia-limited cultures resulted in a rapid loss of GS activity as measured by either the gamma-glutamyl transferase or forward assay method with cells or extracts. No similar activity losses occurred for urease, glutamate dehydrogenase, or pyruvate kinase. The GS activity loss was not prevented by the addition of chloramphenicol and rifampin. The GS activity could be recovered by washing or incubating cells in buffer or by the addition of snake venom phosphodiesterase to cell extracts. Manganese inhibited the GS activity (forward assay) of untreated cells but stimulated the GS activity in ammonia-treated cells. Alanine, glycine, and possibly serine were inhibitory to GS activity. Optimal pH values for GS activity were 7.3 and 7.4 for the forward and gamma-glutamyl transferase assays, respectively. The glutamate dehydrogenase activity was NADPH linked and optimal in the presence of KCl. The data are consistent with an adenylylation-deadenylylation control mechanism for GS activity in S. dextrinosolvens, and the GS pathway is a major route for ammonia assimilation under low environmental ammonia levels. The rapid regulation of the ATP-requiring GS activity may be of ecological importance to this strictly anaerobic ruminal bacterium.     

Enzymes of ammonia assimilation in Rhizobium leguminosarum bacteroids.

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-exeglutarate 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,     

Covalent modification of bacterial glutamine synthetase: physiological significance

Summary Stadtman, Holzer and their colleagues (reviewed in Stadtman and Ginsburg 1974) demonstrated that the enzyme glutamine synthetase (GS) [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] is covalently modified by adenylylation in a variety of bacterial genera and that the modification is reversible. These studies further indicated that adenylylated GS is the less active form in vitro. To assess the physiological significance of adenylylation of GS we have determined the growth defects of mutant strains (glnE) of S. typhimurium that are unable to modify GS and we have determined the basis for these growth defects. The glnE strains, which lack GS adenylyl transferase activity (ATP: [L-glutamate: ammonia ligase (ADP-forming)] adenylyltransferase, EC 2.7.7.42), show a large growth defect specifically upon shift from a nitrogen-limited growth medium to medium containing excess ammonium (NH₄ ⁺). The growth defect appears to be due to very high catalytic activity of GS after shift, which lowers the intracellular glutamate pool to 10% that under preshift conditions. Consistent with this view, recovery of a rapid growth rate on NH₄ ⁺ is accompanied by an increase in the glutamate pool. The glnE strains have normal ATP pools after shift. They synthesize very large amounts of glutamine and excrete glutamine into the medium, but excess glutamine does not seem to inhibit growth. We hypothesize that a major function for adenylylation of bacterial GS is to protect the cellular glutamate pool upon shift to NH₄ ⁺-excess conditions and thereby to allow rapid growth.     

A Metabolic Control Analysis of the Glutamine Synthetase/Glutamate Synthase Cycle in Isolated Barley (Hordeum vulgare L.) Chloroplasts

Ammonia assimilation in chloroplasts occurs via the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. To determine the extent to which these enzymes contribute to the control of ammonia assimilation, a metabolic control analysis was performed on isolated barley (Hordeum vulgare L.) leaf chloroplasts. Pathway flux was measured polarographically as ammonium-plus-2-oxoglutarate-plus-glutamine-dependent O2 evolution in illuminated chloroplasts. Enzyme activity was modulated by titration with specific, irreversible inhibitors of GS (phosphinothricin) and GOGAT (azaserine). Flux control coefficients (CJ0E0) were determined (a) by differentiation of best-fit hyperbolic curves of the data sets (flux versus enzyme activity), and (b) from estimates of the deviation indices (D/[prime]E0). Both analyses gave similar values for the coefficients. The control coefficient for GS was relatively high and the value did not change significantly with changes in 2-oxoglutarate concentration (C/0E0 = 0.58 at 5 mM 2-oxoglutarate and 0.40 at 20 mM 2-oxoglutarate). The control coefficient for GOGAT decreased with decreasing glutamine concentrations, from 0.76 at 20 mM glutamine to 0.19 at 10 mM glutamine. Thus, at high concentrations of glutamine, GOGAT exerts a major control over flux with a significant contribution also from GS. At lower concentrations of glutamine, however, GOGAT exerts far less control over pathway flux.     

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.     

Salt stress affects glutamine synthetase activity and mRNA accumulation on potato plants in an organ-dependent manner

Ammonium assimilation into glutamine and glutamate is vital for plant growth as these are precursors for almost all nitrogenous compounds. Ammonium can be assimilated onto nitrogenous organic compounds by the concerted action of two enzymes that compose the glutamine synthetase (GS, EC 6.3.1.2) – glutamate synthase (Fd-GOGAT, EC 1.4.7.1; NADH–GOGAT, EC 1.4.1.14) cycle. Ammonium may also be directly incorporated into glutamate by the glutamate dehydrogenase (GDH, EC 1.4.1.2) aminating reaction. However, as GDH reversibly deaminates glutamate, its physiological role in vivo remains controversial. Potato has been classified as moderately tolerant to salinity. Potato GS is encoded by a small multigene family which is differentially regulated in an organ and age-dependent way. In this study, the effect of increasing concentrations of salinity in the soil in GS activity and gene-specific mRNA accumulation levels were studied on potato leaves and roots, as well as the biochemical parameters protein, chlorophyll, lipid peroxidation and proline levels, in order to evaluate the severity of the imposed stress. The data obtained suggests that when potato plants are subjected to salt stress, increased ammonium assimilation occurs in roots, due to an increased GS accumulation, along with a decreased assimilation in leaves. Regarding GS gene-specific mRNA accumulation, an organ-dependent response was also observed that contributes for the detected alteration in the ammonium assimilatory metabolism. This response may be a key feature for future genetic manipulations in order to increase crop productivity in salty soils. The possible contribution of GDH for ammonia assimilation was also investigated.