The cloning and nucleotide sequence of cDNA for an amplified glutamine synthetase gene from the Chinese hamster.

The nucleotide sequence for a glutamine synthetase (GS) mRNA from gene-amplified Chinese hamster (CHO) cells was determined from recombinant cDNA clones obtained from both pBR322 and lambda gt10 libraries and by primer extension. The sequence obtained contains about 1400 bp corresponding to a minor species of mRNA terminated by a poly A sequence. The mRNA contains 146 nucleotides of 5'-noncoding region, 1119 bp of coding sequence, and 108 bp of 3'-noncoding sequence with a 32 bp poly(A) tail. The polyadenylation site used shows little homology with efficient polyadenylation sites, but has considerable complementarity with U4 RNA. The predicted amino acid sequence, starting from an initiation codon with the preferred sequence surrounding it, indicates that Chinese hamster GS has high homology with published bovine brain GS peptides and enabled an ordering of these peptides. There is homology between the mammalian GS enzymes and glutamine synthetases obtained from plants and cyanobacteria but no obvious homology between the CHO cell GS sequence and that of other ATP hydrolysing enzymes.     

Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence

Glutamine synthetase is encoded by the glnA gene of Escherichia coli and catalyzes the formation of glutamine from ATP, glutamate, and ammonia. A 1922-base pair fragment from a cDNA containing the glnA structural gene for E. coli glutamine synthetase has been sequenced. An open reading frame of 1404 base pairs encodes a protein of 468 amino acid residues with a calculated molecular weight of 51,814. With few exceptions, the amino acid sequence deduced from the DNA sequence agreed very well with the amino acid sequences of several peptides reported previously. The secondary structure predicted for the E. coli enzyme has approximately 36% of the residues in alpha-helices which is in agreement with calculations of approximately 39% based on optical rotatory dispersion data. Comparison of the amino acid sequences of glutamine synthetase from E. coli (468 amino acids) and Anabaena (473 amino acids) (Turner, N. E., Robinson, S. T., and Haselkorn, R. (1983) Nature 306, 337-342) indicates that 260 amino acids are identical and 80 are of the same type (polar or nonpolar) when aligned for maximum homology. Several homologous regions of these two enzymes exist, including the sites of adenylylation and oxidative modification, but the regulation of each enzyme is different.     

Bacterial type I glutamine synthetase of the rifamycin SV producing actinomycete, Amycolatopsis mediterranei U32, is the only enzyme responsible for glutamine synthesis under physiological conditions

The structural gene for glutamine synthetase, glnA, from Amycolatopsis mediterranei U32 was cloned via screening a genomic library using the analog gene from Streptomyces coelicolor. The clone was functionally verified by complementing for glutamine requirement of an Escherichia coli glnA null mutant under the control of a lac promoter. Sequence analysis showed an open reading frame encoding a protein of 466 amino acid residues. The deduced amino acid sequence bears significant homologies to other bacterial type I glutamine synthetases, specifically, 71% and 72% identical to the enzymes of S. coelicolor and Mycobacterium tuberculosis, respectively. Disruption of this glnA gene in A. mediterranei U32 led to glutamine auxotrophy with no detectable glutamine synthetase activity in vivo. In contrast, the cloned glnA^＋ gene can complement for both phenotypes in trans. It thus suggested that in A. mediterranei U32, the glnA gene encoding glutamine synthetase is uniquely responsible for in vivo glutamine synthesis under our laboratory defined physiological conditions.     

Mg2+ is bound to glutamine synthetase extracted from bovine or ovine brain in the presence of L-methionine-S-sulfoximine phosphate

Purified glutamine synthetase from bovine or ovine brain had no tightly bound Mn2+. By extraction of bovine or ovine brain glutamine synthetase in the presence of L-Met-S-sulfoximine phosphate and ADP in metal ion-free water and 0.1 M KCl, only endogenously bound divalent cations were trapped on the enzyme. Enzyme complexes isolated by immunoprecipitation contained less than 0.05 Mn2+ and 1.5 +/- 0.2 Mg2+ per subunit. Without inactive complex formation, the enzyme immunoprecipitated from extracts contained undetectable Mn2+ (less than 0.01 eq per subunit) and 0.1-2.0 eq of Mg2+ per subunit. Direct binding measurements showed that the purified bovine brain enzyme contained two divalent cations bound at the active site of each subunit. Thus, although either Mg2+ or Mn2+ supports enzyme activity in vitro, Mg2+ rather than Mn2+ appears to be bound to brain glutamine synthetase in vivo.     

Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum.

We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial glnA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon. This glnA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B. japonicum strain.     

Purification, properties and quaternary structure of glutamine synthetase from Chlorella]

A highly purified preparation of glutamine synthetase from chlorella grown on a medium containing nitrate as a sole source of nitrogen, was isolated and characterized by disc-electrophoresis and analytical ultracentrifugation. The N-terminal amino acid of glutamine synthetase is glycine. The molecular weight of glutamine synthetase is 32.000; its activity in the presence of Mg2+ was 150 mkmol o-phosphate per min per mg protein. The molecular weight of subunits of the enzyme, equal to 53.000 was determined by disc-electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate. Electron microscopy of negatively contrasted enzyme preparations revealed 6 subunits in the enzyme molecule, arranged in a point symmetry group 32.     

Inactivation of pea seed glutamine synthetase by the toxin, tabtoxinine-beta-lactam

Glutamine synthetase of plants is the physiological target of tabtoxinine-beta-lactam, a toxin produced by several disease-causing pathovars of Pseudomonas syringae. This toxin, a unique amino acid, is an active site-directed, irreversible inhibitor of glutamine synthetase from pea. ATP is required for inactivation. Neither ADP, AMP, nor adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) supports inactivation. Adenyl-5'-yl imidophosphate (AMP-PNP) is slowly hydrolyzed by glutamine synthetase to produce adenyl-5'-yl phosphoramidate (AMP-PN) and inorganic phosphate as identified by 31P NMR spectroscopic analysis. AMP-PNP also supports a slow inactivation of glutamine synthetase by tabtoxinine-beta-lactam. These data are consistent with gamma-phosphate transfer being involved in the inactivation. Completely inactivated glutamine synthetase has 0.9 mumol of toxin bound/mumol of subunit. One mumol of ATP is bound per mumol of subunit of glutamine synthetase in the absence of either the toxin or another active site-directed inhibitor, methionine sulfoximine; whereas, a 2nd mumol of either [alpha- or gamma-32P]ATP is bound per mumol of subunit when glutamine synthetase is incubated in the presence of either toxin or methionine sulfoximine until all enzyme activity is lost. These data suggest that the gamma-phosphate hydrolyzed from ATP during inactivation remains with the enzyme-inhibitor complex, as well as the ADP. The open chain form, tabtoxinine, was neither a reversible nor an irreversible inhibitor of glutamine synthetase, suggesting that the beta-lactam ring is necessary for inhibition. The inactivation of glutamine synthetase with tabtoxinine-beta-lactam is pseudo-first-order when done in buffer containing 15% (v/v) ethylene glycol. The rate constant for this reaction is 3 X 10(-2) S-1, and the Ki for the toxin is 1 mM. Removal of the ethylene glycol from the buffer allows the reaction to proceed in a non-first-order manner with the apparent rate constant decreasing with time. As the enzyme is inactivated in these conditions, the binding affinity for the toxin appears to decrease, while the Km observed for glutamate does not change.     

Genetic basis for tissue isozymes of glutamine synthetase in elasmobranchs

Tissue-specific isozymes of glutamine synthetase are present in elasmobranchs. A larger isozyme occurs in tissues in which the enzyme is localized in mitochondria (liver, kidney) whereas a smaller form occurs in tissues in which it is cytosolic (brain, spleen, etc.). The nucleotide sequence of spiny dogfish shark (Squalus acanthias) liver glutamine synthetase mRNA, derived from its cDNA, shows there are two in-frame initiation codons (AUG) at the N-terminus which will account for the size differences between the two isozymes. Initiation at the up-stream and down-stream sites would yield peptides of 45,406 and 41,869 mol. wts. representing the precursor of the mitochondrial isozyme and the cytosolic isozyme, respectively. The additional N-terminal 29 amino acids present in the mitochondrial isozyme precursor contains two putative cleavage sites based on the Arg-X-(Phe,Ile,Leu) motif. The predicted two-step processing would remove 14 of the 29 N-terminal amino acids. These 14 amino acids can be predicted to form a very strong amphipathic mitochondrial targeting signal. Their removal would yield a mature peptide of 43,680 mol. wt. The calculated mol. wts. based on the derived amino acid sequence are therefore in good agreement with previous estimates of an approximately 1.5–2-kDa difference between the M_rs of the mitochondrial and cytosolic isozymes. A model for the evolution of the mitochondrial targeting of glutamine synthetase in vertebrates is proposed. Correspondence to: J.W. CampbellThe nucleotide sequence reported will appear in GenBank under accession number U04617     

Rapid transfer of oxygens from inorganic phosphate to glutamine catalyzed by Escherichia coli glutamine synthetase.

Measurements are reported on certain isotopic fluxes during the net conversion of glutamine, ADP and Pi to glutamate, NH3, and ATP by Escherichia coli glutamine synthetase (adenylylated form, Mn2+ activated) in presence of a hexokinase/glucose trap to remove the ATP formed during the reaction. The results show that the transfer of oxygens from Pi to glutamine is the most rapid of the measured isotopic interchanges, over five oxygens from Pi being transferred to glutamine for each glutamate formed by net reaction. Under similar conditions, the oxygen transfer from Pi to glutamate, was stimulated somewhat by an increase in the glutamate concentration but inhibited by an increase in the ammonia concentration. The enzyme from brain or peas did not show the rapid transfer of 18O from Pi to glutamine shown by the E. coli enzyme. Deductions are also made from the data about the availability of the oxygens of gamma-carboxyl of bound glutamate for reaction. The most logical explanation of the results with the E. coli enzyme is that the gamma-carboxyl group of bound glutamate has sufficient rotational freedom so that under conditions of rapid substrate interconversion either carboxylate oxygen can participate in the reaction. The results with the pea enzyme are consistent with hindered rotation of the gamma-care additional findings make likely a relative order of certain catalytic steps for the E. coli enzyme as follows: ATP release less than NH3 release less than glutamate release less than substrate interconversion less than glutamine release and Pi release and glutamate release less than ADP release.     

Nucleotide sequence of the glutamine synthetase gene (glnA) and its upstream region from Bacillus cereus

We have determined the complete nucleotide sequence of a 2.4 kb chromosomal EcoT22I-NspV fragment, containing the Bacillus cereus glnA gene (structural gene of glutamine synthetase). The deduced amino acid sequence indicates that the glutamine synthetase subunit consists of 444 amino acid residues (50,063 Da). Comparisons are made with reported amino acid sequences of glutamine synthetases from other bacteria. Upstrem of glnA we found an open reading frame of 129 codons (ORF129) preceded by the consensus sequence for a typical promoter. Maxicell experiments showed two polypeptide bands, with molecular weights in good agreement with that of glutamine synthetase and that of ORF129, in addition to vector-coded protein. It is possible that the product of this open reading frame upstream of glnA has a regulatory role in glutamine synthetase expression.     

Multiple molecular forms of glutamine synthetase in carp muscles]

Cytoplasmic and mitochondrial molecular forms of glutamine synthetase (CE 6.3.1.2) have been isolated from the carp muscle with purification degree of 100 and 165 times and output 9.0%. It is established that the temperature optimum of the cytoplasmic form activity is 30 degrees C and that of mitochondrial one--20 degrees C; the pH optimum for the both molecular forms is 6.0 and 8.2. The optimal ratio [Me2+] : [ATP] for the isolated form is 2:1; Km (seeming) of the cytoplasmic form in the presence of Mg2+ is 6.0 mM for glutamate, 0.035 for ammonium, for ATP 0.5 and 0.7 for magnesium ions; these values for the mitochondrial form are: 14.3, 0.048, 1.0 and 0.8 mM, respectively. Activity of the both glutamine synthetases with Mg2+ ions is almost by 50% higher than that of glutamine synthetases with Mn2+ ions. Seasonal regularities of the synthesis of molecular glutamine synthetase forms have been established in vivo. Cytoplasmic form is present in the muscles all year round, while mitochondrial one only in winter at low temperature of the environment and fish starvation. Differences in properties and seasonal character of synthesis of molecular glutamine synthetase forms in carp muscles are a result of diversity of their functional role.     

Subunit interaction in unadenylylated glutamine synthetase from Escherichia coli. Evidence from methionine sulfoximine inhibition studies

Although glutamine synthetase from Escherichia coli is composed of 12 identical subunits, there is no evidence that homologous subunit interactions occur in fully unadenylylated or fully adenylylated enzyme. Meister and co-workers (Manning, J. M., Moore, S., Rowe, W. B., and Meister, A. (1969) Biochemistry 8, 2681-2685) have shown that L-methionine-S-sulfoximine, one of the four diastereomers of methionine sulfoximine, preferentially inhibits glutamine synthetase irreversibly in the presence of ATP, due to the formation of tightly bound products, ADP, and methionine sulfoximine phosphate. Using highly purified unadenylylated glutamine synthetase and the two resolved diastereomers of L-methionine-S,R-sulfoximine, we have studied both the kinetics of glutamine synthetase inactivation in the presence of excess methionine sulfoximine and ATP, and the binding of methionine sulfoximine to the enzyme. The results reveal that (a) the apparent first order rate constant of irreversible inactivation by the S isomer decreases progressively from the expected first order rate, indicating that an inactivated subunit retards the reactivity of its neighboring subunits toward methionine sulfoximine and ATP; (b) the R isomer does not inactivate glutamine synthetase irreversibly in the presence of ATP; however, the R isomer is capable of protecting the enzyme temporarily from the irreversible inhibition by the S isomer; and (c) the binding of the S isomer monitored by changes in protein fluorescence exhibits an apparent negative cooperative binding isotherm, whereas the R isomer yields an apparent positive cooperative pattern.     

Formylglycinamide ribonucleotide synthetase from Escherichia coli: cloning, sequencing, overproduction, isolation, and characterization

The purL gene of Escherichia coli encoding the enzyme formylglycinamidine ribonucleotide (FGAM) synthetase which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and MgATP to FGAM, glutamate, ADP, and Pi has been cloned and sequenced. The mature protein, as deduced by the structural gene sequence, contains 1628 amino acids and has a calculated Mr of 141,418. Comparison of the purL control region to other pur loci control regions reveals a common region of dyad symmetry which may be the binding site for the "putative" repressor protein. Construction of an overproducing strain permitted purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of glutamine binding domain sequences (Ebbole & Zalkin, 1987) confirm the amino acid sequence deduced from the gene sequence. The purified protein exhibits glutaminase activity of 0.02% the normal turnover, and NH3 can replace glutamine as a nitrogen donor with a Km = 1 M and a turnover of 3 min-1 (2% glutamine turnover). The enzyme forms an isolable (1:1) complex with glutamine: t1/2 is 22 min at 4 degrees C. This isolated complex is not chemically competent to complete turnover when FGAR and ATP are added, demonstrating that ammonia and glutamine are not covalently bound as a thiohemiaminal available to complete the chemical conversion to FGAM. hydroxylamine trapping experiments indicate that glutamine is bound covalently to the enzyme as a thiol ester. Initial velocity and dead-end inhibition kinetic studies on FGAM synthetase are most consistent with a sequential mechanism in which glutamine binds followed by rapid equilibrium binding of MgATP and then FGAR. Incubation of [18O]FGAR with enzyme, ATP, and glutamine results in quantitative transfer of the 18O to Pi.     

Immunochemical evidence for glutamine-mediated degradation of glutamine synthetase in cultured Chinese hamster cells.

The specific activity of glutamine synthetase in cultured Chinese hamster cells is inversely related to the concentration of glutamine in the surrounding solution. Enzyme specific activity increases 8- to 10-fold when glutamine is removed from serum-free F12 growth media. The induction of glutamine synthetase activity occurs only after glutamine removal and not after the removal of other amino acids (methionine, leucine, or isoleucine). The analysis of the glutamine-mediated decrease in glutamine synthetase activity has been simplified by the finding that depression proceeds in nutrient-free buffered saline solution (141 mM NaCl, 5.4 mM KCl and 30 mM Tricine (pH 7.4). Under these conditions, 0.1 mM cyanide blocks glutamine-mediated depression. The cyanide inhibition is reversed by the addition of 1.0 mM glucose which suggests that ATP is required for depression. Glutamine-mediated depression is temperature-dependent, occurring between 25 and 45 degrees with an optimum rate at 37 degrees. Studies of the time course of induction and depression as a function of glutamine concentration suggest that glutamine regulates the rate at which the enzyme is either modified or degraded. We have employed an antibody prepared against homogeneous Chinese hamster liver glutamine synthetase to measure the amount of glutamine synthetase protein in extracts of cells containing induced or depressed levels of enzyme activity. A highly sensitive immunoprecipitation procedure enables quantitation of nanogram amounts of glutamine synthetase protein. Glutamine synthetase in cell extracts containing induced levels of enzyme activity possesses the same molecular specific activity (ratio of activity to antigenicity) as homogeneous Chinese hamster liver glutamine synthetase. The molecular specific activity of glutamine synthetase is almost the same in extracts of cells with depressed levels of enzyme obtained by growth for short (2 hours) and long (24 hours) times in the presence of glutamine. These data suggest that glutamine-mediated depression of glutamine synthetase results from degradation of enzyme molecules.     

Inactivation of gamma-glutamylcysteine synthetase, but not of glutamine synthetase, by S-sulfocysteine and S-sulfohomocysteine

The reactions catalyzed by gamma-glutamylcysteine synthetase and glutamine synthetase are thought to proceed via enzyme-bound gamma-glutamyl phosphate intermediates. We investigated the possibility that S-sulfocysteine and S-sulfohomocysteine might act as analogs of gamma-glutamyl phosphate or of the associated putative tetrahedral intermediates. The D- and L-enantiomers of S-sulfocysteine and S-sulfohomocysteine were found to rapidly inactivate rat kidney gamma-glutamylcysteine synthetase but to be reversible inhibitors of sheep brain glutamine synthetase. Inactivation of gamma-glutamylcysteine synthetase does not require ATP and is associated with noncovalent binding of close to 1 mol of inactivator/mol of enzyme. The findings indicate that the S-sulfo amino acids are transition-state analogs, and that binding of S-sulfo amino acid to the enzyme induces formation of a very stable enzyme-inactivator complex. The data suggest that stabilization of the enzyme-inactivator complex results from interactions involving the sulfenyl sulfur atom of the S-sulfo amino acid and the active site thiol group of the enzyme.     

Interaction of substrates with glutamine synthetase after limited proteolysis

Previous studies [Dautry-Varsat, A., Cohen, G. N., & Stadtman, E.R. (1979) J. Biol. Chem. 254, 3124-3128; Lei, M., Aebi, U., Heidner, E. G., & Eisenberg, D. (1979) J. Biol. Chem. 254, 3129-3134] have shown that Escherichia coli glutamine synthetase (GS) can be cleaved by proteases to form a limited digestion species called nicked glutamine synthetase (GS). The present study gives the amino acid sequence of the protease-sensitive region of glutamine synthetase. The present study also shows that GS is enzymatically active, but this activity is low compared to the activity of GS. The apparent Michaelis constant value for glutamate was 90 mM for GS as compared to 3 mM for GS, while the Michaelis constant values for ATP were similar for GS and GS*. The dissociation constant values for ATP, as determined by intrinsic fluorescence measurements, were similar for GS and GS*. Glutamate decreased the dissociation constant value of ATP for GS because of synergism between the two binding sites; glutamate did not decrease the dissociation constant value of ATP for GS*. The glutamate analogue methionine sulfoximine bound very tightly to GS and inactivated the enzyme in the presence of ATP. Methionine sulfoximine did not appear to bind to GS* and did not inactivate GS* in the presence of ATP. The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine bound to GS and inactivated the enzyme by forming a covalent bond with it. Glutamate accelerated this inactivation because of the synergism between the ATP and glutamate binding sites of GS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamine synthetase activity and glutamine content in brain: modulation by NMDA receptors and nitric oxide

Acute intoxication with large doses of ammonia leads to rapid death. The main mechanism for ammonia elimination in brain is its reaction with glutamate to form glutamine. This reaction is catalyzed by glutamine synthetase and consumes ATP. In the course of studies on the molecular mechanism of acute ammonia toxicity, we have found that glutamine synthetase activity and glutamine content in brain are modulated by NMDA receptors and nitric oxide. The main findings can be summarized as follows.Blocking NMDA receptors prevents ammonia-induced depletion of brain ATP and death of rats but not the increase in brain glutamine, indicating that ammonia toxicity is not due to increased activity of glutamine synthetase or formation of glutamine but to excessive activation of NMDA receptors.Blocking NMDA receptors in vivo increases glutamine synthetase activity and glutamine content in brain, indicating that tonic activation of NMDA receptors maintains a tonic inhibition of glutamine synthetase.Blocking NMDA receptors in vivo increases the activity of glutamine synthetase assayed in vitro, indicating that increased activity is due to a covalent modification of the enzyme. Nitric oxide inhibits glutamine synthetase, indicating that the covalent modification that inhibits glutamine synthetase is a nitrosylation or a nitration.Inhibition of nitric oxide synthase increases the activity of glutamine synthetase, indicating that the covalent modification is reversible and it must be an enzyme that denitrosylate or denitrate glutamine synthetase.NMDA mediated activation of nitric oxide synthase is responsible only for part of the tonic inhibition of glutamine synthetase. Other sources of nitric oxide are also contributing to this tonic inhibition.Glutamine synthetase is not working at maximum rate in brain and its activity may be increased pharmacologically by manipulating NMDA receptors or nitric oxide content. This may be useful, for example, to increase ammonia detoxification in brain in hyperammonemic situations.