Recombinant Glutamine Synthetase Research Articles

Regulation of glutamine synthetase activity by transcriptional and posttranslational modifications negatively influences ganoderic acid biosynthesis in Ganoderma lucidum.

Glutamine synthetase (GS), a central nitrogen metabolic enzyme, plays important roles in the nitrogen regulation network and secondary metabolism in fungi. However, the mechanisms by which external nitrogen sources regulate fungal GS activity have not been determined. Here, we found that GS activity was inhibited under nitrate conditions in Ganoderma lucidum. By constructing gs-silenced strains and adding 1 mM GS inhibitor to inhibit GS activity, we found that a decrease in GS activity led to a decrease in ganoderic acid biosynthesis. The transcription of gs increased approximately five fold under nitrate conditions compared with that under ammonia. Electrophoretic mobility shift and yeast one-hybrid assay showed that gs was transcriptionally regulated by AreA. Although both gs expression and GS protein content increased under nitrate conditions, the GS activity still decreased. Treatment of recombinant GS with SIN-1 (protein nitration donor) resulted in a strengthened nitration accompanied by a 71% decrease in recombinant GS activity. Furthermore, intracellular GS could be nitrated from mycelia cultivated under nitrate conditions. These results indicated that GS activity could be inhibited by NO-mediated protein nitration. Our findings provide the first insight into the role of transcriptional and posttranslational regulation of GS activity in regulating secondary metabolism in fungi.

Staphylococcus aureus grown in anaerobic conditions exhibits elevated glutamine biosynthesis and biofilm units.

The enormous spread of Staphylococcus aureus infections through biofilms is a major concern in hospital-acquired infections. Biofilm formation by S. aureus on any surface is facilitated by adjusting its redox status. This organism is a facultative anaerobe shift more towards reductive conditions by enhancing nitrogen metabolism where glutamine synthesis plays a key role. Glutamine is synthesized by glutamine synthetase (GS) encoded by the glnA gene. The gene was amplified by PCR from the chromosomal DNA of S. aureus, sequenced (HQ329146.1), and cloned. The pure recombinant GS exhibited Km of 11.06 ± 0.05 mmol·L-1 for glutamate and 2.4 ± 0.03 mmol·L-1 for ATP. The glnA gene sequence showed a high degree of variability with its human counterpart, while it was highly conserved in bacteria. Structural analysis revealed that the GS structure of S. aureus showed close homology with other Gram-positive bacteria and exhibited a high degree of variation with Escherichia coli GS. In the present study, we observed the increased presence of GS activity in multidrug-resistant strains of S. aureus with elevated biofilm units, grown in brain heart infusion broth; among them methicillin-resistant strains S. aureus LMV 3, 4, and 5 showed higher biofilm units. All these results explain the important role of glutamine biosynthesis with elevated biofilm units in the pathogenesis of S. aureus.

Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages.

Glutamine synthetase (GS) generates glutamine from glutamate and controls the release of inflammatory mediators. In macrophages, GS activity, driven by IL10, associates to the acquisition of M2‐like functions. Conditional deletion of GS in macrophages inhibits metastasis by boosting the formation of anti‐tumor, M1‐like, tumor‐associated macrophages (TAMs). From this basis, we evaluated the pharmacological potential of GS inhibitors in targeting metastasis, identifying glufosinate as a specific human GS inhibitor. Glufosinate was tested in both cultured macrophages and on mice bearing metastatic lung, skin and breast cancer. We found that glufosinate rewires macrophages toward an M1‐like phenotype both at the primary tumor and metastatic site, countering immunosuppression and promoting vessel sprouting. This was also accompanied to a reduction in cancer cell intravasation and extravasation, leading to synchronous and metachronous metastasis growth inhibition, but no effects on primary tumor growth. Glufosinate treatment was well‐tolerated, without liver and brain toxicity, nor hematopoietic defects. These results identify GS as a druggable enzyme to rewire macrophage functions and highlight the potential of targeting metabolic checkpoints in macrophages to treat cancer metastasis.

FRI113 - Recombinant glutamine synthetase (AM-535): a novel therapeutic approach for the treatment of hyperammonaemia and its deleterious effects on multiple systems

FRI113 - Recombinant glutamine synthetase (AM-535): a novel therapeutic approach for the treatment of hyperammonaemia and its deleterious effects on multiple systems

Recombinant glutamine synthetase reduces ammonia and hepatic fibrosis in in vitro and in vivo models of non-alchoholic fatty liver disease

Recombinant glutamine synthetase reduces ammonia and hepatic fibrosis in in vitro and in vivo models of non-alchoholic fatty liver disease

The role of glutamine synthetase in the pathogenesis of Neisseria meningitidis

Neisseria meningitidisis a cause of meningitis and severe sepsis. A moonlighting protein is a protein with the ability to perform additional task(s) alongside its recognised function. These proteins have been identified in both prokaryotic and eukaryotic cells and they represent a highly conserved subset of proteins that typically are either metabolic pathway-associated enzymes or act as molecular chaperones. To explore the role of GlnA in the pathogenesis of meningococcal disease, the encoding gene designated NMB0359, was amplified from the wild-type meningococcal strain MC58 and cloned into the pQE-30 expression vector. Recombinant glutamine synthetase (rGlnA) was expressed in E. coli and purified by immobilised metal affinity chromatograph. Rabbit antisera was raised against purified rGlnA (RαGlnA) and used to investigate the localisation of GlnA at the cell surface. Attempts were also made to generate glnA knockout and complemented strains of wild-type N. meningitidis. rGlnA was successfully purified from E. coli cell lysates under native conditions. A highly immuno-reactive band of the expected size (52 kDa) was observed when rGlnA immunoblot was probed with RαGlnA. GlnA could be detected on the surface of wild-type encapsulated N. meningitidis MC58 using whole-cell enzyme linked immunosorbent assay (ELISA). Surface localisation of GlnA indicates that it may be a moonlighting protein carrying out function(s) at the cell surface. Future work will investigate possible moonlighting functions which may include adhesion to host cells and proteins, regulation of the host immune response, and contribution to bacterial virulence.

Mechanistic Basis for ATP-Dependent Inhibition of Glutamine Synthetase by Tabtoxinine-β-lactam.

Tabtoxinine-β-lactam (TβL), also known as wildfire toxin, is a time- and ATP-dependent inhibitor of glutamine synthetase produced by plant pathogenic strains of Pseudomonas syringae. Here we demonstrate that recombinant glutamine synthetase from Escherichia coli phosphorylates the C3-hydroxyl group of the TβL 3-(S)-hydroxy-β-lactam (3-HβL) warhead. Phosphorylation of TβL generates a stable, noncovalent enzyme-ADP-inhibitor complex that resembles the glutamine synthetase tetrahedral transition state. The TβL β-lactam ring remains intact during enzyme inhibition, making TβL mechanistically distinct from traditional β-lactam antibiotics such as penicillin. Our findings could enable the design of new 3-HβL transition state inhibitors targeting enzymes in the ATP-dependent carboxylate-amine ligase superfamily with broad therapeutic potential in many disease areas.

Bisphosphonic acids as effective inhibitors of Mycobacterium tuberculosis glutamine synthetase

Inhibition of glutamine synthetase (GS) is one of the most promising strategies for the discovery of novel drugs against tuberculosis. Forty-three bisphosphonic and bis-H-phosphinic acids of various scaffolds, bearing aromatic substituents, were screened against recombinant GS from Mycobacterium tuberculosis. Most of the studied compounds exhibited activities in micromolar range, with N-(3,5-dichlorophenyl)-2-aminoethylidenebisphoshonic acid, N-(3,5-difluorophenyl)-2-aminoethylidene-bisphoshonic acid and N-(3,4-dichlorophenyl)-1-hydroxy-1,1-ethanebisphosphonic acid showing the highest potency with kinetic parameters similar to the reference compound – L-methionine-S-sulfoximine. Moreover, these inhibitors were found to be much more effective against pathogen enzyme than against the human ortholog. Thus, with the bone-targeting properties of the bisphosphonate compounds in mind, this activity/selectivity profile makes these compounds attractive agents for the treatment of bone tuberculosis.

Molecular cloning and characterization of glutamine synthetase, a tegumental protein from Schistosoma japonicum

Glutamine synthetase catalyzes the synthesis of glutamine, providing nitrogen for the production of purines, pyrimidines, amino acids, and other compounds required in many pivotal cellular events. Herein, a full-length cDNA encoding Schistosoma japonicum glutamine synthetase (SjGS) was isolated from 21-day schistosomes. The entire open reading frame of SjGS contains a 1,095-bp coding region corresponding to 364 amino acids with a calculated molecular weight of 40.7 kDa. NCBIP blast shows that the putative amino acid of SjGS contains a classic β-grasp domain and a catalytic domain of glutamine synthetase. The relative mRNA expression of SjGS was evaluated in 7-, 13-, 21-, 28-, 35-, and 42-day worms of S. japonicum in the final host and higher expression at day 21, and 42 worms were observed. This protein was also detected in worm extracts using Western blot. Immunofluorescence studies indicated that the SjGS protein was mainly distributed on tegument and parenchyma in 28-day adult worms. The recombinant glutamine synthetase with a molecular weight of 45 kDa was expressed in Escherichia coli and purified in its active form. The enzyme activity of the recombinant protein was 3.30 ± 0.67 U.μg-1. The enzyme activity was highly stable over a wide range of pH (6-9) and temperature (25-40 °C) under physiological conditions. The transcription of SjGS was upregulated in praziquantel-treated worms at 2-, 4-, and 24-h posttreatment compared with the untreated control. As a first step towards the clarification of the role of glutamine synthetase in schistosome species, we have cloned and characterized cDNAs encoding SjGS in S. japonicum, and the data presented suggest that SjGS is an important molecule in the development of the schistosome.

Production of glutamine synthetase in Escherichia coli using SUMO fusion partner and application to l-glutamine synthesis

l-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine industry is expected. High efficiency of l-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v) lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS–PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel nitrilo-triacetic acid (Ni–NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 121 mg recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l l-Gln was detected by high pressure liquid chromatography (HPLC) or thin-layer chromatography. Thus, the application of SUMO technology to the expression and purification of GS potentially could be employed for the industrial production of l-Gln.

Characterization of protein acyltransferase function of recombinant purified GlnA1 from Mycobacterium tuberculosis: A moon lighting property

The protein acetyltransferase (MTAase) function of glutamine synthetase of Mycobacterium smegmatis was established earlier. In this paper, studies were undertaken to examine MTAase function of recombinant glutamine synthetase (rGlnA1) of Mycobacterium tuberculosis, which showed >80% similarity with M. smegmatis GlnA. The specificity of MTAase to several acyl derivative of coumarins was examined. The results clearly indicated that MTAase exhibited differential specificities to several acyloxycoumarins. Further, MTAase was also found capable of transferring propionyl and butyryl groups from propoxy and butoxy derivatives of 4-methylcoumarin. These observations characterized MTAase in general as a protein acyltransferase. MTAase catalyzed acetylation of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model acetoxy coumarin was confirmed by MALDI-TOF-MS as well as western blot analysis using acetylated lysine polyclonal antibody. In order to validate the active site of rGlnA1 for TAase activity, effect of DAMC and L-methionine-S-sulfoximine (MSO) on GS and TAase activity of rGlnA1 were studied. The results indicated that the active sites of GS and TAase were found different. Acetyl CoA, a universal biological acetyl group donor, was also found to be a substrate for MTAase. These results appropriately characterize glutamine synthetase of Mtb exhibiting transacylase action as a moonlighting protein.

Recombinant Glutamine Synthetase (GS) from C. glutamicum Existed as Both Hexamers & Dedocamers and C-terminal His-tag Enhanced Inclusion Bodies Formation in E. coli

In order to investigate the effect of his-tag on glutamine synthetase (GS, EC 6.3.1.2) from Corynebacterium glutamicum, recombinant Escherichia coli strains overexpressing GSIM, HGSIM (GS fused with N-terminal his-tag), GSIMH (GS fused with C-terminal his-tag), and HGSIMH (GS fused with N-terminal & C-terminal his-tags) were constructed, respectively. Under similar expression conditions, GSIM and HGSIM were partially solubly expressed; no soluble GSIMH and HGSIMH were observed, based on the result of SDS-PAGE. Gel filtration of purified soluble HGSIM showed that hexamers and dedocamers coexisted in the quaternary structure of GS from C. glutamicum. Combined this result with the analysis of two GS crystal structure models, we hypothesized that C-terminal residues participated in GS folding after translation on ribosome. After the folding process, C-terminal residues were released again and exposed to solvent. Fused C-terminal his-tag interrupted the GS to fold into its correct conformation so that inclusion bodies formed.

Establishment of Glutamine Synthetase of Mycobacterium smegmatis as a Protein Acetyltransferase utilizing Polyphenolic Acetates as the Acetyl Group Donors

Acetoxy Drug: Protein Transacetylase (TAase) mediating the transfer of acetyl group(s) from polyphenolic acetates (PA) to certain functional proteins in mammalian cells was identified by our earlier investigations. TAase activity was characterized in the cell lysates of Mycobacterium smegmatis and the purified protein was found to have M(r) 58,000. TAase catalysed protein acetylation by a model acetoxy drug 7,8-diacetoxy-4-methylcoumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with an anti-acetyllysine antibody. The specificity of the TAase of M. smegmatis (MTAase) to various acetoxycoumarins was found to be in the order DAMC > 7-AMC > 6-AMC > 4-AC > 3-AC > ABP. Also, the N-terminal sequence of purified MTAase was found to perfectly match with glutamine synthetase (GS) of M. smegmatis. The identity of MTAase with GS was confirmed by the observation that the purified MTAase as well as the purified recombinant GS exhibited all the properties of GS. The finding that purified Escherichia coli GS was found to have substantial TAase activity highlighted the TAase function of GS in other bacteria. These results conclusively established for the first time the protein acetyltransferase function of GS of M. smegmatis.

Mn2+ enhances theanine-forming activity of recombinant glutamine synthetase from Bacillus subtilis in Escherichia coli

Bacillus subtilis glutamine synthetase (GS) was highly expressed (about 86% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-glnA, which was induced by 0.4 mM IPTG in LB medium, and maximal theanine-forming activity of the recombinant GS induced in LB is 6.4 U/mg at a series concentration (0–100 mM) of Mn2+ at optimal pH 7.5. In order to get GS with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9-A (details are described in “Materials and methods”) and 0.1% (w/v) lactose were selected as culture medium and inducer respectively. Recombinant GS was also highly expressed (84% of total protein) and totally soluble in M9-A and the specific activity of the recombinant GS is 6.2 U/mg which is approximate to that (6.4 U/mg) induced in LB in the presence of 10 mM Mn2+ at optimal pH 7.5. The activity is markedly higher activated by Mn2+ than that by other nine bivalent cations. Furthermore, M9-B (5 μM Mn2+ was added into M9-A) was used to culture the recombinant strain and theanine-forming activity of the recombinant GS induced in M9-B was improved 20% (up to 7.6 U/mg). Finally, theanine production experiment coupled with yeast fermentation system was carried out in a 1.0 ml reaction system with 0.1 mg crude GS from M9-B or M9-A, and the yield of theanine were 15.3 and 13.1 g/L by paper chromatography and HPLC, respectively.

Cloning and Expression ofPseudomonas taetrolensY-30 Gene Encoding Glutamine Synthetase: An Enzyme Available for Theanine Production by Coupled Fermentation with Energy Transfer

Glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 can form theanine from glutamic acid and ethylamine in a mixture where yeast fermentation of sugar is coupled for ATP regeneration (coupled fermentation with energy transfer). From a genomic DNA library of P. taetrolens Y-30, a clone containing 6 kbp insertional DNA fragment was selected by the PCR screening technique with specific oligonucleotide primers for the GS gene. The fragment had an open reading frame of the GS gene encoding a protein of 468 amino acids (molecular mass, 52 kDa). The deduced amino acid sequence showed a significant homology with that of P. syringae pv. tomato GS (97%), and all the amino acid residues were fully conserved, which concern with catalytic activity in other bacterial GS. A tyrosine residue for adenylylation of GS was also found, and in vivo adenylylation was confirmed in P. taetrolens Y-30. The isolated GS gene was ligated into an expression vector (pET21a), and expressed in Escherichia coli AD494 (DE3). The enzyme productivity in the expression system was 30-fold higher than that in P. taetrolens Y-30. Recombinant GS had the same properties as those of unnadenylylated intrinsic GS, and formed theanine in the mixture of coupled fermentation with energy transfer.

Cloning and expression of mycobacterial glutamine synthetase gene in Escherichia coli

Extracellular glutamine synthetase (GS) is one of the prominent proteins secreted by pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium bovis. Non-pathogenic species like Mycobacterium smegmatis and Mycobacterium phlei do not secrete this protein. GS has been proposed to play an indispensable role in intracellular survival of pathogenic mycobacteria. In this study, the structural gene for extracellular glutamine synthetase of M. tuberculosis was PCR amplified and expressed as fusion protein with hexahistidine residues in Escherichia coli. Expression of GS in E. coli under transcriptional regulation of T5 promoter yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in presence of 8 M urea and the enzyme was purified to homogeneity under denaturing conditions using nitrilotriacetic acid (Ni–NTA) affinity chromatography. The denatured protein was renatured by gradual removal of the urea while immobilized on (Ni–NTA) column. The yield of purified recombinant glutamine synthetase was 40 mg/L. The purified recombinant enzyme was obtained in highly active state having specific activity of 200 U/mg protein. This is the first report describing cloning and expression of mycobacterial glutamine synthetase gene in E. coli.

Immunoassay for Serum Glutamine Synthetase in Serum: Development, Reference Values, and Preliminary Study in Dementias

Glutamine synthetase (GS) is a ubiquitous enzyme present at high concentrations in liver, muscle, kidney, and brain (1). GS in the brain is astrocyte specific, is involved in the detoxification of ammonia and glutamate, and is overexpressed after brain injury (2). Monomeric GS protein was found in 38 of 39 cerebrospinal fluid samples obtained from Alzheimer disease (AD) patients (3), and the concentration of GS in the lumbar cerebral spinal fluid of AD patients was increased significantly but nonspecifically (4). GS has also been reported in serum (4). Markers for AD have poor sensitivity and specificity for overt or preclinical AD (5)(6). We have developed an ELISA that is rapid, highly sensitive, and GS specific. This ELISA, together with the S100B and neuron-specific enolase (NSE) ELISAs, was used to retrospectively assess serum GS in healthy controls, AD patients, and other dementia patients. Recombinant human GS (rhGS) cDNA was purchased from ATCC (ATCC No. 409071). The full-length rhGS open reading frame was obtained by PCR and subcloning in pET28a ( Nde l/ Xho I). The construct included a polyhistidine tag at the N-terminal domain of the rhGS open reading frame and no extra sequence at the C-terminal domains. The protein was expressed in E. coli BL21 (DE3) (7). We prepared extracts and solubilized products as described (8). Affinity purification was performed by nickel-nitrilotriacetic acid chromatography. Female Balb/c mice (7–8 weeks of age) were immunized by subcutaneous injection of 50 μg of rhGS emulsified in complete Freund’s adjuvant. An intraperitoneal booster injection was given 3 weeks later (50 μg in incomplete Freund’s adjuvant) and then at 2- to 3-week intervals (three times or more) with a final injection of 50 μg of antigen in buffer via intraperitoneal route 3 days before cell fusion. Hybridoma cultures were screened by ELISA, and …

Beta-Glutamate as a substrate for glutamine synthetase.

The conversion of beta-glutamate to beta-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 alpha-glutamate. Recombinant GS from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus were considerably more selective for alpha-glutamate than beta-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 beta-glutamate were much smaller than rates with the alpha-isomer. These results are discussed in light of the observation that beta-glutamate is accumulated as an osmolyte in many archaea while beta-glutamine (produced by glutamine synthetase) is used as an osmolyte only in M. portucalensis.

The plastidic glutamine synthetase activity is directly modulated by means of redox change at two unique cysteine residues

Two cDNA clones for glutamine synthetase (GS), Clgln1 and Clgln2, were isolated from a Canavalia lineata cDNA library constructed with poly(A)+ RNA from the mature plant leaves. From a comparison of the primary structures, Clgln1 was judged to encode a cytosolic isoform and Clgln2 to encode a plastidic isoform. It was revealed by those sequence comparisons that the two cysteine residues (Cys-306 and Cys-371) of Clgln2 (chloroplastic GS encoding gene) were substituted by alanine and serine in the Clgln1 clone (cytosolic GS encoding gene), respectively. These cysteine substitutions were found between chloroplastic GS (GS2) and cytosolic GS (GS1) sequences of all plants. These unique cysteine residues may account for the specific susceptibility of the plastidic GS by sulfhydryl reagents. To investigate this assumption the two additional cysteine residues of GS2 were mutated combinatorially and the resulting recombinant GS proteins as well as the two wild-type cytosolic GS and chloroplastic GS were examined in vitro. Only GS2, of the wild-type forms, was significantly activated by dithiotreitol. Moreover, the mutant form, mutated at both of the two additional cysteine residues, was not activated by the reductant, but the mutant forms mutated at only one of the two were also activated.

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.