林肯链霉菌谷氨酰胺合成酶的酶学性质

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.     

Purification and characterization of glutamine synthetase of Pseudomonas taetrolens Y-30: an enzyme usable for production of theanine by coupling with the alcoholic fermentation system of baker's yeast

Concentrated cell-extract of Pseudomonas taetrolens Y-30, isolated as a methylamine-assimilating organism, formed gamma-glutamylethylamide (theanine) from glutamic acid and ethylamine in a mixture containing the alcoholic fermentation system of baker's yeast for ATP-regeneration. Glutamine synthetase (GS), probably responsible for theanine formation, was isolated from the extract of the organism grown on a medium containing 1% methylamine, 1% glycerol, 0.5% yeast extract, and 0.2% polypepton as carbon and nitrogen sources. The molecular mass was estimated to be 660 kDa by gel filtration and 55 kDa by SDS-polyacrylamide gel electrophoresis, suggesting that Ps. taetrolens Y-30 GS consists of 12 identical subunits. The enzyme required Mg2+ or Mn2+ for its activity. Under the standard reaction condition for glutamine formation (pH 8.0 with 30 mM Mg2+), GS showed 7% and 1% reactivity toward methylamine and ethylamine respectively of that to ammonia. Reactivity to the alkylamines varied with optimum pH of the reaction in response to divalent cation in the mixture: pH 11.0 was the optimum for the Mg2+ -dependent reaction with ethylamine, and pH 8.5 was the optimum for the Mn2+ -dependent reaction. In a mixture of an optimum reaction condition with 1000 mM ethylamine (at pH 8.5 with 3 mM Mn2+), reactivity increased up to 7% of the reactivity to ammonia in the standard reaction condition. The isolated GS formed theanine in the mixture with the yeast fermentation system.     

Expression,purification, and characterization of recombinant mangrove glutamine synthetase

To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was ~40 kDa. Optimal conditions for biosynthetic activity were found to be 35 °C at pH 7.5. The Mg²⁺-dependent biosynthetic activity was strongly inhibited by Ni²⁺, Zn²⁺, and Al³⁺, whereas was enhanced by Co²⁺. The apparent K _m values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, and 2.54 mM for ATP, 2.80 mM for NH₄ ⁺ respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions.     

The role of specific cations in regulation of cyanobacterial glutamine synthetase

Purified glutamine synthetase from the cyanobacterium Anabaena cylindrica required a divalent cation for activity. Maximum biosynthetic activity required Mg2+ (25 mM when supplied alone). Co2+ and Mn2+ each supported up to 20% of this activity; 12 other cations tested were ineffective. At 2.5 - 10 mM Mg2+, 0.1 mM Co2+ or ethylene glycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) stimulated GS activity to maximum rates; other divalent cations (particularly Mn2+) inhibited Mg2+-dependent activity. At 5 mM Mg2+ the Kappm for NH+4 (0.05 mM) was 20-fold lower than at 25 mM Mg2+; added Co2+ did not markedly alter this low Km for NH+4; this could be physiologically important.     

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.     

GLUTAMINE SYNTHETASE IN MARINE ALGAE: NEW SURPRISES FROM AN OLD ENZYME

Glutamine synthetase (GS), which catalyzes the formation of glutamine from ammonium and glutamate in the presence of ATP, is encoded by three distinct gene families: GSI, GSII, and GSIII. Genes encoding GSI are found in the Bacteria and Archaea, whereas GSII genes are found in eukaryotes and a few species of Bacteria. Members of the third family, GSIII, have been described from a limited number of bacteria; however, recent biochemical and molecular data suggest that this type of enzyme is broadly distributed among the algae. Peptide fragments obtained from GS purified from the marine diatom Skeletonema costatum (Greville) Cleve are 77% identical to a partial sequence of GSIII from Chaetoceros compressum Lauder, which permits the unambiguous assignment of the biochemically characterized enzyme to the GSIII gene family. The N-terminal sequence was 43% identical to the GSIII-like enzyme purified from the haptophyte Emiliania huxleyi (Lohm.) Hay et Miller and several residues were conserved among bacterial and eukaryotic GSIII enzymes. The presence of genes encoding GSIII in diatoms and haptophytes indicates that this enzyme family is more broadly distributed in eukaryotes than previously suspected.     

Molecular evolution of glutamine synthetase II: Phylogenetic evidence of a non-endosymbiotic gene transfer event early in plant evolution

Background  

Glutamine synthetase (GS) is essential for ammonium assimilation and the biosynthesis of glutamine. The three GS gene families (GSI, GSII, and GSIII) are represented in both prokaryotic and eukaryotic organisms. In this study, we examined the evolutionary relationship of GSII from eubacterial and eukaryotic lineages and present robust phylogenetic evidence that GSII was transferred from γ-Proteobacteria (Eubacteria) to the Chloroplastida.     

A previously unrecognized glutamine synthetase expressed in Klebsiella pneumoniae from the glnT locus of Rhizobium leguminosarum

Summary Using glnT DNA of Rhizobium meliloti as a hybridization probe we identified a R. leguminosarum biovar phaseoli (R. l. phaseoli) locus (glnT) expressing a glutamine synthetase activity in Klebsiella pneumoniae. A 2.2 kb DNA fragment from R. l. phaseoli was cloned to give plasmid pMW5a, which shows interspecific complementation of a K. pneumoniae glnA mutant. The cloned sequence did not show cross-hybridization to glnA or glnII, the genes coding for two glutamine synthetase isozymes of Rhizobium spp. While in previous reports on glnT of R. meliloti and Agrobacterium tumefaciens no glutamine synthetase activity was detected, we do find activity with the glnT locus of R. l. phaseoli. The glutamine synthetase (GSIII) activity expressed in a K. pneumoniae glnA strain from pMW5a shows a ratio of biosynthetic to transferase activity 10³-fold higher than that observed for GSI or GSII. GSIII is similar in molecular weight and heat stability to GSI.     

Regulation and properties of glutamine synthetase purified from Bacillus cereus

The glutamine synthetase from Bacillus cereus IFO 3131 was purified to homogeneity. The enzyme is a dodecamer with a molecular weight of approximately 600,000, and its subunit molecular weight is 50,000. Both Mg2+ and Mn2+ activated the enzyme as to the biosynthesis of L-glutamine, but, unlike in the case of the E. coli enzyme, the Mg2+-dependent activity was stimulated by the addition of Mn2+. The highest activity was obtained when 20 mM Mg2+ and 0.5 mM Mn2+ were added to the assay mixture. For each set of optimal assay conditions, the apparent Km values for glutamate, ammonia and a divalent cation X ATP complex were 1.03, 0.34, and 0.40 mM (Mn2+: ATP = 1: 1); 14.0, 0.47, and 0.91 mM (Mg2+: ATP = 4: 1); and 9.09, 0.45, and 0.77 mM (Mg2+: Mn2+: ATP = 4: 0.2: 1), respectively. At each optimum pH, the Vmax values for these reactions were 6.1 (Mn2+-dependent), 7.4 (Mg2+-dependent), and 12.9 (Mg2+ plus Mn2+-dependent) mumoles per min per mg protein, respectively. Mg2+-dependent glutamine synthetase activity was inhibited by the addition of AMP or glutamine; however, this inhibitory effect was suppressed in the case of the Mg2+ plus Mn2+-dependent reaction. These results suggest that the activity of the B. cereus glutamine synthetase is regulated by both the intracellular concentration and the ratio of Mn2+/Mg2+ in vivo. Also in the present investigation, a potent glutamine synthetase inhibitor(s) was detected in crude extracts from B. cereus.     

Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting.     

Low- and high-activity forms of glutamine synthetase from Rhodospirillum rubrum: sensitivity to feed-back effectors and activation of the low-activity form.

Glutamine synthetase from Rhodospirillum rubrum can be isolated in two forms, with low and high activity, respectively, depending on the concentration of combined nitrogen in the medium before harvest. The two forms have been studied with respect to their dependence on Mn2+ and Mg2+ in both the transferase and the biosynthetic assay. There is no difference in pH optimum between the forms in the biosynthetic assay. In addition the pH-optima for the two cations studied are very close, 7.4 (Mg2+) and 7.2 (Mn2+). It also shows that the activity of the low-activity form is higher than that of the high-activity form in the Mn(2+)-dependent biosynthetic assay. The two forms of Rsp. rubrum glutamine synthetase have also been studied with respect to their sensitivity towards feed-back effectors. In the transferase assay both forms are inhibited to essentially the same degree by alanine, glycine, histidine, AMP, CTP and UTP, CTP being the most effective of the nucleotides and of the amino acids alanine causes the highest inhibition. In the biosynthetic assay these effectors show different degrees of inhibition on the two different forms; the high-activity form being the most sensitive. The results are discussed in relation to properties of glutamine synthetase from Escherichia coli and other phototropic bacteria in which regulation of glutamine synthetase is known to be due to adenylylation. It is also shown that the low-activity form of Rsp. rubrum glutamine synthetase can be activated in crude extracts in a reaction that is inhibited by glutamine.     

Regulation and biochemical characterization of the glutamine synthetase of azotobacter vinelandii

We have investigated the regulation of the activity and synthesis of the glutamine synthetase (l-glutamate:ammonia ligase (ADP-forming), EC (6.3.1.2) of Azotobacter vinelandii. Synthesis of the enzyme was not repressed by NH+4 and/or a number of amino acids in the growth medium; however, biosynthetic activity was rapidly lost through adenylylation in response to ammonium ion. The enzyme could be prepared as a 'relaxed, divalent-cation-free form which was catalytically inactive. The 'taut', active form could be restored with 1-5 mM Mg2+, Mn2+, Ca2+ or CO2+ and taut-vs.-relaxed difference spectra unique to each divalent cation were generated. Mg2+ and CO2+ each supported biosynthetic catalysis, but with different substrate Km and Vmax values. L-Alanine, glycine and L-aspartate were the most potent of several inhibitors of the biosynthetic and the gamma-glutamyl transferase activities; only aspartate and AMP behaved differentially toward glutamine synthetase adenylylation state: the more highly adenylylated enzyme was more severely affected. Any two of alanine, glycine or AMP showed cumulative inhibition, while the inhibitory effects of groups of three effectors were not cumulative. The Co2+-supported biosynthetic activity of Al vinelandii glutamine synthetase was markedly less sensitive to inhibition my glycine and alanine and was stimulated up to 50% by 1-10 mM aspartate.     

The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli.

The structural gene (glnA) encoding the glutamine synthetase (GS) of the extremely thermophilic eubacterium Thermotoga maritima has been cloned on a 6.0 kb HindIII DNA fragment. Sequencing of the region containing the glnA gene (1444 bp) showed an ORF encoding a polypeptide (439 residues) with an estimated mass of 50,088 Da, which shared significant homology with the GSI sequences of other Bacteria (Escherichia coli, Bacillus subtilis) and Archaea (Pyrococcus woesei, Sulfolobus solfataricus). The T. maritima glnA gene was expressed in E. coli, as shown by the ability to complement a glnA lesion in the glutamine-auxotrophic strain ET8051. The recombinant GS has been partially characterized with respect to the temperature dependence of enzyme activity, molecular mass and mode of regulation. The molecular mass of the Thermotoga GS (590,000 Da), estimated by gel filtration, was compatible with a dodecameric composition for the holoenzyme, as expected for a glutamine synthetase of the GSI type. Comparison of the amino acid sequence of T. maritima GS with those from thermophilic and mesophilic micro-organisms failed to detect any obvious features directly related to thermal stability.     

Purification and properties of glutamine synthetase from the non-N2-fixing cyanobacterium Phormidium laminosum.

Soluble glutamine synthetase activity (L-glutamate:ammonia ligase, ADP forming, EC 6.3.1.2) was purified to electrophoretic homogeneity from the filamentous non-N2-fixing cyanobacterium Phormidium laminosum (OH-1-p.Cl1) by using conventional purification procedures in the absence of stabilizing ligands. The pure enzyme showed a specific activity of 152 mumol of gamma-glutamylhydroxamate formed.min-1 (transferase activity), which corresponded to 4.4 mumol of Pi released.min-1 (biosynthetic activity). The relative molecular mass of the native enzyme was 602 kilodaltons and was composed of 12 identically sized subunits of 52 kilodaltons. Biosynthetic activity required the presence of Mg2+ as an essential activator, although Co2+ and Zn2+ were partially effective. The kinetics of activation by Mg2+, Co2+, and Zn2+ were sigmoidal, and concentrations required for half-maximal activity were 18 mM (h = 2.2), 6.3 mM (h = 5.6), and 6.3 mM (h = 2.45), respectively. However, transferase activity required Mn2+ (Ka = 3.5 microM), Cu2+, Co2+, or Mg2+ being less effective. The substrate affinities calculated for L-Glu, ammonium, ATP, L-Gln, and hydroxylamine were 15, 0.4, 1.9 (h = 0.75), 14, and 4.1 mM, respectively. Optimal pH and temperature were 7.2 and 55 degrees C for biosynthetic activity and 7.5 and 45 degrees C for transferase activity. The biosynthetic reaction mechanism proceeded according to an ordered three-reactant system, the binding order being ammonium, L-Glu, and ATP. The presence of Mn2+ or Mg2+ drastically affected the thermostability of transferase and biosynthetic activities. Heat inactivation of biosynthetic activity in the presence of Mn2+ obeyed first-order kinetics, with an Ea of 76.8 kcal (ca. 321 kJ) mol-1. Gly, L-Asp, L-Ala, L-Ser and, with lower efficiency, L-Lys and L-Met, L-Lys, and L-Glu inhibited only transferase activity. No cumulative inhibition was observed when mixtures of amino acids were used. Biosynthetic activity was inhibited by AMP (Ki= 7 mM), ADP (Ki= 2.3 mM), p-hydroxymercuribenzoate (Ki= 25 microM), and L-methionine-D, L-sulfoximine (Ki= 2 microM). The enzyme was not activated in vitro by chemically reduced Anabaena thioredoxin. This is the first report of glutamine synthetase activity purified from a filamentous non-N2-fixing cyanobacterium.     

Relationships between nitrogenase,glutamine synthetase,glutamine, and energy charge in Azotobacter vinelandii

When continuous cultures of Azotobacter vinelandii were supplied with ammonium or nitrate in amounts, which just repressed nitrogenase synthesis completely, both the intracellular glutamine level and the degree of adenylylation of the glutamine synthetase (GS) increased only slightly (from 0.45–0.50 mM and from 2 to 3 respectively), while the total GS level remained unaffected. Higher amounts of ammonium additionally inhibited the nitrogenase activity, caused a strong rise in the intracellular glutamine concentration and adenylylation of the GS, but caused no change in the ATP/ADP ratio. These results are considered as evidence that in A. vinelandii the regulation of nitrogenase synthesis is not linked to the adenylylation state of the GS and to the intracellular glutamine level, and that the inhibition of the nitrogenase activity as a consequence of a high extracellular ammonium level is not mediated via a change in the energy charge.Abbreviations GS glutamine synthetase - GS-S(Mg) Mg²⁺ dependent synthetic activity of GS - GS-T(Mn) Mn²⁺ dependent transferase activity of GS     

Identification and characterization of three forms of glutamine synthetase unique to Rhizobia

Glutamine synthetase (GS) activities of Rhizobia were chromatographically resolved into three distinct forms, GSI, GSII, and GSIII on DEAE cellulose, being eluted with 0.3M, 0.5M and 0.8M KCl, respectively. GSIII was the major form inR. leguminosarum andR. phaseoli. InR. meliloti, however, GSI was the major form. The three forms of GS were also distinguished on the basis of (a) rapid heat inactivation of GSII, (b) insensitivity of GSI to inhibitors, (c) marked inhibition of GSII by thymidine, and (d) inability of Zn⁺⁺ to inhibit GSIII. The three forms of GS are also distinct molecular entities and are unique to Rhizobia.     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

Multiple mechanisms by which glutamine synthetase levels are controlled in murine tissue culture cells

We report the isolation of a complimentary DNA (cDNA) clone encoding glutamine synthetase, derived from a population of methionine sulfoxime-resistant mouse GF1 fibroblasts. When GF1 cells are incubated for 48 h in the presence of the glucocorticoid hormone dexamethasone, the specific activity of glutamine synthetase (GS), assayed as glutamyltransferase activity, increases by threefold. Based on dot hybridization analysis, hormonal treatment also produces a similar increase in the level of GS mRNA. When GF1 cells or mouse Neuro 2A neuroblastoma cells are transferred from medium containing 4 mM glutamine to glutamine-free medium, glutamyltransferase activity increases by at least fivefold. However, the presence or absence or glutamine in the medium does not affect the relative level of glutamine synthetase mRNA in either cell line. With both GF1 and Neuro 2A cells, the half-time for the decline in glutamine synthetase enzyme activity on addition of glutamine to the medium is approximately 1.5 h. This rapid decline, coupled with the lack of effect of glutamine on the level of GS messenger RNA in Neuro 2A cells, renders it unlikely that neural cells alter glutamine synthetase levels in response to glutamine by a biosynthetic mechanism, as suggested by previous authors [L. Lacoste, K.D. Chaudhary, and J. Lapointe (1982) J. Neurochem. 39, 78-85].     

Air-breathing catfish, Clarias batrachus upregulates glutamine synthetase and carbamyl phosphate synthetase III during exposure to high external ammonia

We assessed the possible upregulation of glutamine synthetase (GS) and typical 'fish type' carbamyl phosphate synthetase III (CPS III) in detoxification of ammonia in different tissues of the walking catfish (Clarias batrachus) during exposure to 25 mM NH(4)Cl for 7 days. Exogenous ammonia led to an increase in ammonia and urea concentrations in different tissues. The results revealed the presence of relatively high levels of GS activity in the brain, liver and kidney, unexpectedly, also in the muscle, and even higher levels in the intestine and stomach. Exposure to high external ammonia (HEA) caused significant increase of activities of GS, CPS III and CPS I-like enzymes, accompanied with the upregulation of GS and CPS III enzyme proteins in different tissues. Exposure to HEA also led to a sharp rise of plasma cortisol level, suggesting being one of the primary causes of upregulation of GS and CPS III enzymes activity. Liver perfusion experiments further revealed that exposure to HEA enhances the capacity of trapping ammonia to glutamine and urea by the liver of walking catfish. These results suggest that the upregulation of GS and CPS III activity in walking catfish during exposure to HEA plays critical roles to ameliorate the toxic ammonia to glutamine, and also to urea via the induced ornithine-urea cycle possibly through the involvement of cortisol.