Evolutionary relationships of the carbamoylphosphate synthetase genes

Carbamoylphosphate is a common intermediate in the metabolic pathways leading to the biosynthesis of arginine and pyrimidines. The amino acid sequences of all available proteins that catalyze the formation of carbamoylphosphate were retrieved from Genbank and aligned to estimate their mutual phylogenetic relations. In gram-negative bacteria carbamoylphosphate is synthesized by a two-subunit enzyme with glutamiriase and carbamoylphosphate synthetase (CPS) activity, respectively. In gram-positive bacteria and lower eukaryotes this two-subunit CPS has become dedicated to arginine biosynthesis, while in higher eukaryotes the two subunits fused and subsequently lost the glutaminase activity. The CPS dedicated to pyrimidine synthesis is part of a multifunctional enzyme (CPS II), encoding in addition dihydroorotase and aspartate transcarbamoylase. Evidence is presented to strengthen the hypothesis that the two kinas subdomains of all CPS isozymes arose from a duplication of an ancestral gene in the progenote. A further duplication of the entire CPS gene occurred after the divergence of the plants and before the divergence of the fungi from the eukaryotec root, generating the two isoenzymes involved in either the synthesis of arginine or that of pyrimidines. The mutation rate was found to be five- to tenfold higher after the duplication than before, probably reflecting optimization of the enzymes for their newly acquired specialized function. We hypothesize that this duplication arose from a need for metabolic channeling for pyrimidine biosynthesis as it was accompanied by the tagging of the CPS gene with the genes for dihydroorotase and aspartate transcarbamoylase, and as the duplication occurred independently also in gram-positive bacteria. Analysis of the exon-intron organization of the two kinase subdomains in CPS I and II suggests that ancient exons may have comprised approx. 19 amino acids, in accordance with the prediction of the intron-early theory. Correspondence to: M.J.B. van den Hoff     

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: Phylogenetic implications

Summary The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%–49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.     

Recent developments on the regulation and structure of glutamine synthetase enzymes from selected bacterial groups

Abstract: The structure of glutamine synthetase (GS) enzymes from diverse bacterial groups fall into three distinct classes. GSI is the typical bacterial GS, GSII is similar to the eukaryotic GS and is found together with GSI in plant symbionts and Streptomyces , while GSIII has been found in two unrelated anaerobic rumen bacteria. In most cases, the structural gene for GS enzyme is regulated in response to nitrogen. However, different regulatory mechanisms, to ensure optimal utilization of nitrogen substrates, control the GS enzyme in each class.     

Purification and characterization of glutamine synthetase from Rhodomicrobium vannielii

Abstract Glutamine synthetase (GS) from the purple non-sulfur bacterium Rhodomicrobium vannielii has been purified to electrophoretic homogeneity by affinity chromatography. Molecular weight and catalytic properties of the enzy,e are similar to those described for other species of Rhodospirillaceae. However, the enzyme from this organism appears to be antigenically different from the glutamine synthetases of other species of Rhodospirillaceae studied.     

Immunogold localization of glutamine synthetase in soybean leaves,roots, and nodules

Summary Immunogold labelling was used to detect the cellular and sub-cellular distribution of glutamine synthetase (GS) in nodulatedGlycine max var. maple arrow. The protein was detected in thin sections of tissue embedded in LR white acrylic resin by employing two polyclonal antibody preparations, one active chloroplastic GS, the other against the cytosolic form of the enzyme. In the mature leaf tissue, GS was visualized only in the chloroplasts, exclusively within the stroma matrix; in the root cortical tissue, the enzyme was distributed homogenously in the cytosol but with a slight preferential localization associated with certain endomembranes, whereas in the root nodules both cytosolic and plastidial compartments were labelled in infected and uninfected cells. Particular to the infected cells, the bacteroids' inner matrix reacted slightly to the GS antibody and a strong signal was preferentially localized on the bacteroids' outer envelope membranes. In general, GS was more concentrated in nodules as estimated by gold particle distribution, whether in the cytosol, plastids or on the bacteroid envelope membranes, than in either root tissue or leaf tissue. Although the cytoplasmic labelling density in nodules was similar in uninfected and infected cells, certain structural features in the latter (abundant cytosol, numerous GS-positive bacteroids and GS-reactive proplastids) contribute to a more enzyme-rich type than its uninfected counterpart.Abbrevation GS glutamine synthetase     

Metabolic characteristics of recombinant Chinese hamster ovary cells expressing glutamine synthetase in presence and absence of glutamine

To elucidate the metabolic characteristics of recombinant CHO cells expressing glutamine synthetase (GS) in the medium with or without glutamine, the concentrations of extra- and intracellular metabolites and the activities of key metabolic enzymes involved in glutamine metabolism pathway were determined. In the absence of glutamine, glutamate was utilized for glutamine synthesis, while the production of ammonia was greatly decreased. In addition, the expression of recombinant protein was increased by 18%. Interestingly, the intracellular glutamine maintained almost constant, independent of the presence of glutamine or not. Activities of glutamate-oxaloacetate aminotransferase (GOT), glutamate-pyruvate aminotransferase (GPT), and glutamate dehydrogenase (GDH) increased in the absence of glutamine. On the other hand, intracellular isocitrate and the activities of its downstream isocitrate dehydrogenase in the TCA cycle increased also. In combination with these two factors, a 8-fold increase in the intracellular α-ketoglutarate was observed in the culture of CHO-GS cells in the medium without glutamine.     

Molecular evolution of duplicate copies of genes encoding cytosolic glutamine synthetase in Pisum sativum

Here, we describe two nearly identical expressed genes for cytosolic glutamine synthetase (GS3A and GS3B) in Pisum sativum L. RFLP mapping data indicates that the GS3A and GS3B genes are separate loci located on different chromosomes. DNA sequencing of the GS3A and GS3B genes revealed that the coding regions are 99% identical with only simple nucleotide substitutions resulting in three amino acid differences. Surprisingly, the non-coding regions (5 non-coding leader, the 11 introns, and 3 non-coding tail) all showed a high degree of identity (96%). In these non-coding regions, 25% of the observed differences between the GS3A and GS3B genes were deletions or duplications. The single difference in the 3 non-coding regions of the GS3A and GS3B genes was a 25 bp duplication of an AU-rich element in the GS3B gene. As the GS3B mRNA accumulates to lower levels than the GS3A gene, we tested whether this sequence which resembles an mRNA instability determinant functioned as such in the context of the GS mRNA. Using the GS3B 3 tail as part of a chimeric gene in transgenic plants, we showed that this AU-rich sequence has little effect on transgene mRNA levels. To determine whether the GS3A/GS3B genes represent a recent duplication, we examined GS3-like genes in genomic DNA of ancient relatives of P. sativum. We observed that several members of the Viceae each contain two genomic DNA fragments homologous to the GS3B gene, suggesting that this is an ancient duplication event. Gene conversion has been invoked as a possible mechanism for maintaining the high level of nucleotide similarity found between the GS3A and GS3B genes. Possible evolutionary reasons for the maintenance of these twin GS genes in pea, and the general duplication of genes for cytosolic GS in all plant species are discussed.     

Purification and properties of glutamine synthetase from the archaebacterium Halobacterium salinarium

Glutamine synthetase (GS) was purified to electrophoretic homogeneity from the halophilic archaebacterium Halobacterium salinarium. The enzyme was purified 300-fold to homogeneity with 30% yield. By gel filtration and SDS gel electrophoresis, it was shown that the enzyme has a native molecular weight of 495,000 and a subunit molecular weight of 62,000. This indicates an octameric quaternary structure. The amino acid composition and the isoelectric point of 4.9 are similar to other GSs. The enzyme shows highest stability in 4 M NaCl or KCl and at temperatures up to 45°C. Lower salt concentrations or higher temperatures lead to rapid and irreversible denaturation. By low concentrations of Mg²⁺ or Mn²⁺, the salt dependence was decreased and the thermostability increased. Mg²⁺ or Mn²⁺ are essential cofactors. The two resulting activities show differences in pH and salt concentrations required for optimal activity, different K _m-values and different sensitivity to inhibition by amino acids. The enzyme is not adenylylated like the GS from some eubacteria but cytidylylated. The covalently bound CMP increases Mn²⁺-and Mg²⁺-dependent activities at a different extent.     

Aminoacyl-tRNA synthetase classes and groups in prokaryotes

Knowledge on the evolution of aminoacyl-tRNA synthetases is crucial to studies on the origins of life. The relationships between the different aminoacyl-tRNA synthetase specificities in prokaryotic organisms are studied in this work. We reconstructed the ancestor sequences and the phylogenetic relationships utilizing the Maximum Likelihood method. The results suggest that in class I the evolution of the N-terminal segment was strongly influenced by the amino acid hydropathy in both domains of prokaryotes. The results for the C-terminal segments of class I were different in the two domains, indicating that its evolution was strongly influenced by the specific types of tRNA modification in each domain. The class II groups in Archaea were more heterogeneous with respect to the hydropathy of amino acids, indicating the interference of other influences. In bacteria, the configuration was also complex but the overall consensual division in two groups was maintained, group IIa forming a single branch with the five hydroapathetic amino acid specificities and group IIb containing the specificities for the moderately hydrophobic together with the hydrophilic amino acids. It is indicated that the aminoacyl-tRNA synthetase in both domains were subjected to different selective forces in diverse parts of the proteins, resulting in complex phylogenetic patterns.     

Glutamine synthetase desensitizes differentiated adipocytes to proinflammatory stimuli by raising intracellular glutamine levels

The role of glutamine synthetase (GS) during adipocyte differentiation is unclear. Here, we assess the impact of GS on the adipocytic response to a proinflammatory challenge at different differentiation stages. GS expression at the late stages of differentiation desensitized mature adipocytes to bacterial lipopolysaccharide (LPS) by increasing intracellular glutamine levels. Furthermore, LPS-activated mature adipocytes were unable to produce inflammatory mediators; LPS sensitivity was rescued following GS inhibition and the associated drop in intracellular glutamine levels. The ability of adipocytes to differentially respond to LPS during differentiation negatively correlates to GS expression and intracellular glutamine levels. Hence, modulation of intracellular glutamine levels by GS expression represents an endogenous mechanism through which mature adipocytes control the inflammatory response.     

Glutamine synthetase and glutamine synthetase-like protein from human brain: purification and comparative characterization

Glutamine synthetase (GS; EC 6.3.1.2), a key enzyme of glutamate metabolism, and another enzyme possessing high hydroxylamine-L-glutamine transferase activity comparable to that of GS and termed GS-like protein (GSLP) were purified from human brain concurrently. In two-dimensional electrophoresis, GS subunits migrate to at least six different positions (44 +/- 1 kDa, pl = 6. 4-6.7), whereas GSLP subunits migrate to at least four different positions (54 +/- 1 kDa, pl = 5.9-6.2). Dependences of enzymatic activity in the transferase reaction on concentrations of Mn(2+) and Mg(2+) for GS and GSLP are different. High immunological cross-reactivity between GS and GSLP was observed in ELISA. Nevertheless, antisera were raised to GS and GSLP, and a method was developed for the separate detection of GS and GSLP in brain extracts by enzyme-chemiluminescent amplified (ECL) immunoblotting. The distribution of GS and GSLP immunoreactivities between soluble protein and crude mitochondrial fractions indicates tighter association with the particulate fraction for GSLP than for GS. The results from activity measurements suggest that the hydroxylamine-L-glutamine transferase activity measured routinely in protein extracts from brain is the sum of GS and GSLP activities. Similarly, immunoreactivity evaluated by ELISA is a sum of immunoreactivities of GS and GSLP. The relative contributions of GS and GSLP to the total immunoreactivity can be evaluated by ECL-immunoblotting.     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Azospirillum brasilense glutamine synthetase: influence of the activating metal ions on the enzyme properties

The kinetic properties of the Mg²⁺-activated and Mn²⁺-activated glutamine synthetase (GS) of Azospirillum brasilense in the biosynthetic reaction were studied. The Mg²⁺-supported and Mn²⁺-supported GSs in an average state of adenylylation varied in pH optimum, maximum activity, saturation functions for ammonium and glutamate, affinity to substrates, and in the Me²⁺-ATP ratio required for the optimal enzyme activity. Seventeen other cations were tested for the maintenance of GS activity. The level of the latter and the kinetic behavior of the GS in A.brasilense is suggested to depend essentially on the concentrations of Mg²⁺, Mn²⁺ and Co²⁺, as well as on their ratio     

Overproduction of alfalfa glutamine synthetase in transgenic tobacco plants

Summary We have obtained transgenic tobacco plants overexpressing the enzyme glutamine synthetase (GS) by fusing an alfalfa GS gene to the cauliflower mosaic virus 35S promotor and integrating it intoNicotiana tabacum var. W38 plants byAgrobacterium tumefaciens mediated gene transfer. The amount of RNA specific to alfalfa GS was about 10 times higher in transgenic tobacco plants than in alfalfa. The alfalfa GS produced by these transgenic plants was identified by Western blotting and represented 5% of total soluble protein in the transformed plants, amounting to a 5-fold increase in specific GS activity and in a 20-fold increase in resistance to the GS inhibitorl-phosphinothricin in vitro. Tissue from GS overproducing plants showed a sevenfold lower amount of free NH₃. The amino acid composition of the plant tissue was not altered significantly by GS overproduction. GS overproducing plants were fertile and grew normally. These data show that a high level of expression of a key metabolic enzyme such as glutamine synthetase does not interfere with growth and fertility of plants.     

Changes in the activity levels of glutamine synthetase, glutaminase and glycogen synthetase in rats subjected to hypoxic stress

 Exposure to high altitude causes loss of body mass and alterations in metabolic processes, especially carbohydrate and protein metabolism. The present study was conducted to elucidate the role of glutamine synthetase, glutaminase and glycogen synthetase under conditions of chronic intermittent hypoxia. Four groups, each consisting of 12 male albino rats (Wistar strain), were exposed to a simulated altitude of 7620 m in a hypobaric chamber for 6 h per day for 1, 7, 14 and 21 days, respectively. Blood haemoglobin, blood glucose, protein levels in the liver, muscle and plasma, glycogen content, and glutaminase, glutamine synthetase and glycogen synthetase activities in liver and muscle were determined in all groups of exposed and in a group of unexposed animals. Food intake and changes in body mass were also monitored. There was a significant reduction in body mass (28–30%) in hypoxia-exposed groups as compared to controls, with a corresponding decrease in food intake. There was rise in blood haemoglobin and plasma protein in response to acclimatisation. Over a three-fold increase in liver glycogen content was observed following 1 day of hypoxic exposure (4.76±0.78 mg·g⁻¹ wet tissue in normal unexposed rats; 15.82±2.30 mg·g⁻¹ wet tissue in rats exposed to hypoxia for 1 day). This returned to normal in later stages of exposure. However, there was no change in glycogen synthetase activity except for a decrease in the 21-days hypoxia-exposed group. There was a slight increase in muscle glycogen content in the 1-day exposed group which declined significantly by 56.5, 50.6 and 42% following 7, 14, and 21 days of exposure, respectively. Muscle glycogen synthetase activity was also decreased following 21 days of exposure. There was an increase in glutaminase activity in the liver and muscle in the 7-, 14- and 21-day exposed groups. Glutamine synthetase activity was higher in the liver in 7- and 14-day exposed groups; this returned to normal following 21 days of exposure. Glutamine synthetase activity in muscle was significantly higher in the 14-day exposed group (4.32 μmol γ-glutamyl hydroxamate formed·g protein⁻¹·min⁻¹) in comparison to normal (1.53 μmol γ-glutamyl hydroxamate formed·g protein⁻¹·min⁻¹); this parameter had decreased by 40% following 21 days of exposure. These results suggest that since no dramatic changes in the levels of protein were observed in the muscle and liver, there is an alteration in glutaminase and glutamine synthetase activity in order to maintain nitrogen metabolism in the initial phase of hypoxic exposure. Received: 30 March 1998 / Revised: 18 November 1998 / Accepted: 25 November 1998