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1.
Patrice Robinson Kelly Neelon Harold J. Schreier Mary F. Roberts 《Applied microbiology》2001,67(10):4458-4463
The conversion of β-glutamate to β-glutamine by archaeal and bacterial glutamine synthetase (GS) enzymes has been examined. The GS from Methanohalophilus portucalensis (which was partially purified) is capable of catalyzing the amidation of this substrate with a rate sevenfold less than the rate obtained with α-glutamate. Recombinant GS from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus were considerably more selective for α-glutamate than β-glutamate as a substrate. All the archaeal enzymes were much less selective than the two bacterial GS (from Escherichia coli and Bacillus subtilis), whose specific activities towards β-glutamate were much smaller than rates with the α-isomer. These results are discussed in light of the observation that β-glutamate is accumulated as an osmolyte in many archaea while β-glutamine (produced by glutamine synthetase) is used as an osmolyte only in M. portucalensis. 相似文献
2.
Neelon K Schreier HJ Meekins H Robinson PM Roberts MF 《Biochimica et biophysica acta》2005,1753(2):164-173
Methanococcus jannaschii accumulates alpha- and beta-glutamate as osmolytes. The effect of these and other solutes on the thermostability of two multisubunit metabolic enzymes from M. jannaschii, aspartate transcarbamoylase catalytic trimer (ATCase C3) and glutamine synthetase (GS), has been measured and compared to solute effects on bacterial mesophilic counterparts in order to explore if osmolytes accumulated by each organism can preferentially stabilize the proteins to thermal unfolding. For both ATCase enzymes and for the B. subtilis GS, the solutes normally accumulated by the organism were very effective in protecting the enzyme from losing activity at high temperatures, although solute effects on loss of secondary structure did not necessarily correlate with this thermoprotection of activity. The recombinant M. jannaschii GS exhibited quite different behavior. The pure enzyme had a thermal unfolding transition with a midpoint temperature (Tm) less than 60 degrees C, well under the growth temperature of the organism (85 degrees C). None of the small molecule solutes tested (including the K+-glutamate isomers accumulated by M. jannaschii) significantly stabilized the protein to incubation at 85 degrees C. Instead, protein-protein interactions, as illustrated by E. coli GroEL or ribosomal protein L2 stabilization of GS, appeared to be the dominant factor in stabilizing this archaeal enzyme at the growth temperature. 相似文献
3.
In vivo NMR studies of the thermophilic archaeon Methanococcus thermolithotrophicus, with sodium formate as the substrate for methanogenesis, were used to monitor formate utilization, methane production, and osmolyte pool synthesis and turnover under different conditions. The rate of formate conversion to CO2 and H2 decreased for cells adapted to higher external NaCl, consistent with the slower doubling times for cells adapted to high external NaCl. However, when cells grown at one NaCl concentration were resuspended at a different NaCl, formate utilization rates increased. Production of methane from 13C pools varied little with external NaCl in nonstressed culture, but showed larger changes when cells were osmotically shocked. In the absence of osmotic stress, all three solutes used for osmotic balance in these cells, l-alpha-glutamate, beta-glutamate, and Nepsilon-acetyl-beta-lysine, had 13C turnover rates that increased with external NaCl concentration. Upon hyperosmotic stress, there was a net synthesis of alpha-glutamate (over a 30-min time-scale) with smaller amounts of beta-glutamate and little if any of the zwitterion Nepsilon-acetyl-beta-lysine. This is a marked contrast to adapted growth in high NaCl where Nepsilon-acetyl-beta-lysine is the dominant osmolyte. Hypoosmotic shock selectively enhanced beta-glutamate and Nepsilon-acetyl-beta-lysine turnover. These results are discussed in terms of the osmoadaptation strategies of M. thermolithotrophicus. 相似文献
4.
Glycine betaine transport in the obligate halophilic archaeon Methanohalophilus portucalensis
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Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe Methanohalophilus portucalensis (strain FDF1), since solute uptake for this class of obligate halophilic methanogenic Archaea has not been examined. Betaine uptake followed a Michaelis-Menten relationship, with an observed K(t) of 23 microM and a V(max) of 8 nmol per min per mg of protein. The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for [(14)C]betaine uptake. The proton-conducting uncoupler 2, 4-dinitrophenol and the ATPase inhibitor N, N-dicyclohexylcarbodiimide both inhibited glycine betaine uptake. Growth of cells in the presence of 500 microM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, alpha-glutamate, beta-glutamine, and N(epsilon)-acetyl-beta-lysine. These investigations demonstrate that this model halophilic methanogen, M. portucalensis strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions. 相似文献
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6.
Krajewski WW Collins R Holmberg-Schiavone L Jones TA Karlberg T Mowbray SL 《Journal of molecular biology》2008,375(1):217-228
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. 相似文献
7.
8.
《Journal of Fermentation and Bioengineering》1989,67(6):395-398
After the addition of ammonia to the culture medium, the concentration of glutamine in B. flavum cells increased in 20 s with a decrease in glutamate. In the subsequent 30 s, the glutamine concentration deceased again with an increase in glutamate. An enzyme system, which consisted of purified glutamine synthetase (GS) and glutamate synthase (GOGAT) with ATP- and NADPH-regenerating systems, was made up to study the functions of the GS/GOGAT pathway: concentrations of the substrates and of the enzymes were decided on according to the intracellular conditions. Changes in the concentrations of amino acids caused by the addition of ammonia to the system were very similar to those of intracellular glutamate and glutamine when ammonia was added to the bacterial culture. The time required for the complete formation of glutamate from 0.5 mM ammonia was about 4-times shorter in the GS/GOGAT system than in the system using purified glutamate dehydrogenase (GDH) and the NADPH-regenerating system. The glutamate synthase reaction in the GS/GOGAT system was inhibited by some amino acids much more markedly than in the standard assay mixture consisting of glutamine, α-ketoglutarate and NADPH. These results gave further evidence elucidating the operation of the GS/GOGAT pathway in ammonia assimilation, and suggested that a reconstructed enzyme system is useful for studying physiological mechanisms. 相似文献
9.
Recent developments on the regulation and structure of glutamine synthetase enzymes from selected bacterial groups 总被引:5,自引:0,他引:5
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. 相似文献
10.
Hirotaka Yamamoto Hidehiko Konno Teiji Yamamoto Kitae Ito Michinao Mizugaki Yuzo Iwasaki 《Journal of neurochemistry》1987,49(2):603-609
Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of gamma-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver. 相似文献
11.
Takuya Ishibashi Hiroya Tomita Yuusuke Yokooji Tatsuya Morikita Bunta Watanabe Jun Hiratake Asako Kishimoto Akiko Kita Kunio Miki Tadayuki Imanaka Haruyuki Atomi 《Extremophiles : life under extreme conditions》2012,16(6):819-828
We have previously reported that the majority of the archaea utilize a novel pathway for coenzyme A biosynthesis (CoA). Bacteria/eukaryotes commonly use pantothenate synthetase and pantothenate kinase to convert pantoate to 4′-phosphopantothenate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, two novel enzymes specific to the archaea, pantoate kinase and phosphopantothenate synthetase, are responsible for this conversion. Here, we examined the enzymatic properties of the archaeal phosphopantothenate synthetase, which catalyzes the ATP-dependent condensation of 4-phosphopantoate and β-alanine. The activation energy of the phosphopantothenate synthetase reaction was 82.3?kJ?mol?1. In terms of substrate specificity toward nucleoside triphosphates, the enzyme displayed a strict preference for ATP. Among several amine substrates, activity was detected with β-alanine, but not with γ-aminobutyrate, glycine nor aspartate. The phosphopantothenate synthetase reaction followed Michaelis–Menten kinetics toward β-alanine, whereas substrate inhibition was observed with 4-phosphopantoate and ATP. Feedback inhibition by CoA/acetyl-CoA and product inhibition by 4′-phosphopantothenate were not observed. By contrast, the other archaeal enzyme pantoate kinase displayed product inhibition by 4-phosphopantoate in a non-competitive manner. Based on our results, we discuss the regulation of CoA biosynthesis in the archaea. 相似文献
12.
Characteristics and Efficiency of Glutamine Production by Coupling of a Bacterial Glutamine Synthetase Reaction with the Alcoholic Fermentation System of Baker’s Yeast
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Shinji Wakisaka Yoshifumi Ohshima Masahiro Ogawa Tatsurokuro Tochikura Takashi Tachiki 《Applied microbiology》1998,64(8):2952-2957
Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker’s yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain. 相似文献
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14.
Mongin AA Hyzinski-García MC Vincent MY Keller RW 《American journal of physiology. Cell physiology》2011,301(4):C814-C822
Here we report and validate a simple method for measuring intracellular activities of glial glutamine synthetase (GS) and glutaminase (GLNase) in intact glial cells. These enzymes are responsible for glutamate and glutamine recycling in the brain, where glutamate and glutamine transport from the blood stream is strongly limited by the blood-brain barrier. The intracellular levels of glutamate and glutamine are dependent on activities of numerous enzymatic processes, including 1) cytosolic production of glutamine from glutamate by GS, 2) production of glutamate from glutamine by GLNase that is primarily localized between mitochondrial membranes, and 3) mitochondrial conversion of glutamate to the tricarboxylic cycle intermediate α-ketoglutarate in the reactions of oxidative deamination and transamination. We measured intracellular activities of GS and GLNase by quantifying enzymatic interconversions of L-[(3)H]glutamate and L-[(3)H]glutamine in cultured rat astrocytes. The intracellular substrate and the products of enzymatic reactions were separated in one step using commercially available anion exchange columns and quantified using a scintillation counter. The involvement of GS and GLNase in the conversion of (3)H-labeled substrates was verified using irreversible pharmacological inhibitors for each of the enzymes and additionally validated by measuring intracellular amino acid levels using an HPLC. Overall, this paper describes optimized conditions and pharmacological controls for measuring GS and GLNase activities in intact glial cells. 相似文献
15.
Goude R Renaud S Bonnassie S Bernard T Blanco C 《Applied and environmental microbiology》2004,70(11):6535-6541
Erwinia chrysanthemi is a phytopathogenic soil enterobacterium closely related to Escherichia coli. Both species respond to hyperosmotic pressure and to external added osmoprotectants in a similar way. Unexpectedly, the pools of endogenous osmolytes show different compositions. Instead of the commonly accumulated glutamate and trehalose, E. chrysanthemi strain 3937 promotes the accumulation of glutamine and alpha-glucosylglycerate, which is a new osmolyte for enterobacteria, together with glutamine. The amounts of the three osmolytes increased with medium osmolarity and were reduced when betaine was provided in the growth medium. Both glutamine and glutamate showed a high rate of turnover, whereas glucosylglycerate stayed stable. In addition, the balance between the osmolytes depended on the osmolality of the medium. Glucosylglycerate and glutamate were the major intracellular compounds in low salt concentrations, whereas glutamine predominated at higher concentrations. Interestingly, the ammonium content of the medium also influenced the pool of osmolytes. During bacterial growth with 1 mM ammonium in stressing conditions, more glucosylglycerate accumulated by far than the other organic solutes. Glucosylglycerate synthesis has been described in some halophilic archaea and bacteria but not as a dominant osmolyte, and its role as an osmolyte in Erwinia chrysanthemi 3937 shows that nonhalophilic bacteria can also use ionic osmolytes. 相似文献
16.
A Metabolic Control Analysis of the Glutamine Synthetase/Glutamate Synthase Cycle in Isolated Barley (Hordeum vulgare L.) Chloroplasts 总被引:3,自引:1,他引:2
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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. 相似文献
17.
林肯链霉菌谷氨酰胺合成酶的酶学性质 总被引:4,自引:0,他引:4
在分离纯化的基础上,报道了pH、温度和金属离子对林肯链霉菌(Streptomyceslincolnensis)Z-512谷氨酸胺合成酶(GS)活力的影响及GS底物专一性的研究结果.在动力学性质的研究中,发现林肯链霉菌GS在生物合成反应系统中,对底物NH_4CI的饱和曲线不遵守米氏方程.Hill作图呈两相曲线.在NH_4CI浓度低的情况下,Hill系数大于1,具有正协同效应;当NH_4CI浓度增加到一定程度时,Hill系数小于1,具有负协同效应.这说明NH_4CI不仅作为林肯链霉菌GS的底物,而且作为一种效应物调节GS的活性.林肯链霉菌GS对底物Glu及ATP的饱和曲线遵守米氏方程.在不同的激活离子存在下,GS对Glu、ATP的Km值也不同. 相似文献
18.
Intact bundle-sheath cells with functional plasmodesmata were isolated from leaves of Zea mays L. cv. Mutin, and the capacity of these cells to synthesize glutamine and glutamate was determined by simulating physiological substrate concentrations in the bathing medium. The results show that glutamine synthetase can operate at full rate in the presence of added 8 mM ATP. At lower concentrations of ATP a higher rate of glutamine synthesis was found in the light than in darkness. Glutamate-synthase activity, on the other hand, was strictly light dependent. It appears that in bundle-sheath cells of maize the nitrate-assimilatory capacities of glutamine synthetase (located mainly in the cytosol) and of glutamate synthase (located in the stroma) are high enough to meet the demands of whole maize leaves.Abbreviations Gln
glutamine
- Glu
glutamate
- GOGAT
glutamate synthase
- GS
glutamine synthetase
- 2-OG
2-oxoglutarate
This work was supported by the Bundesminister für Forschung und Technologie (0319296A). We thank Mr. Bernd Raufeisen for the art work of Fig. 1. 相似文献
19.
Plants of Urtica dioica L., a very nitrophilous species, were grown in a nutrient solution containing either high (100%) or low (2%) nutrient supply. Part of these plants were subjected to a sudden switch from 100% to 2% or vice versa. Plant weight, sugar and organic nitrogen (both soluble and insoluble) and nitrate content were measured during growth. The activities of two nitrogen assimilating enzymes, nitrate reductase (NR) and glutamine synthetase (GS) were determined.
Growth of Urtica dioica was retarded at low nutrient supply. Root growth was limited by another factor than nitrogen. This was shown by a higher protein content. In the first period after a switch from 100% to 2%, redistribution of nitrogen from shoot to root could be demonstrated, and leakage from the root into the nutrient solution. It is suggested that in these conditions GS in the root reacted to this downward flux. Comparison with earlier findings on the less nitrophilous Plantago lanceolata showed that at 100% nutrient supply a correlation occurs between nitrate reduction and glutamine synthetase activity in that plant part which exported reduced nitrogen: the root in P. lanceolata and the shoot in U. dioica. In the importing plant part, glutamine synthetase was influenced by nitrate reduction as well as by imported reduced nitrogen. 相似文献
Growth of Urtica dioica was retarded at low nutrient supply. Root growth was limited by another factor than nitrogen. This was shown by a higher protein content. In the first period after a switch from 100% to 2%, redistribution of nitrogen from shoot to root could be demonstrated, and leakage from the root into the nutrient solution. It is suggested that in these conditions GS in the root reacted to this downward flux. Comparison with earlier findings on the less nitrophilous Plantago lanceolata showed that at 100% nutrient supply a correlation occurs between nitrate reduction and glutamine synthetase activity in that plant part which exported reduced nitrogen: the root in P. lanceolata and the shoot in U. dioica. In the importing plant part, glutamine synthetase was influenced by nitrate reduction as well as by imported reduced nitrogen. 相似文献
20.
Molecular physiology of glutamine and glutamate biosynthesis in developing seedlings of conifers 总被引:4,自引:0,他引:4
Francisco M. Cánovas Francisco R. Cantón Angel GarcíaGutiérrez Fernando Gallardo Remedios Crespillo 《Physiologia plantarum》1998,103(2):287-294
Nitrogen is a limiting factor in tree growth and development. The incorporation of ammonium ions in carbon skeletons is catalyzed by the sequential action of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT). Most studies on nitrogen‐assimilating enzymes have been reported for annual crop plants. Knowledge of these enzymes in woody plants is much more limited, particularly at the molecular level. Here, we review current available information on glutamine/glutamate biosynthesis and chloroplast development in conifers. 相似文献