Structural exploration of glutamine synthetase from Leishmania donovani: Insights from in silico and in vitro analysis.

Glutamine synthetase from L. donovani (LdGS) has been identified as a potential antileishmanial target in our previous report based on biochemical and inhibition studies. With the aim to structurally explore LdGS, systematic in silico and in vitro studies have been employed in the present study to identify amino acids crucial for LdGS mediated catalysis. A comparative analysis with human GS (HsGS) was performed which revealed significant differences in the active site pocket of human and parasite GS enzyme. The important amino acids identified from the in silico analysis of the optimized complexes, were subjected to in silico and in vitro alanine scanning by site directed mutagenesis. The results indicated crucial conserved and non conserved residues required for GS activity. The role of these residues in maintenance of secondary and tertiary structure of GS enzyme was also explored. In silico virtual screening was performed which resulted in the identification of five hits i.e. ZINC83236243, ZINC77319454, ZINC83236244, ZINC83236734 and ZINC83236736, as potential LdGS selective inhibitors. The illustrated structural and functional details of enzyme provides a better understanding of the structural integrity of LdGS and can be further utilized for the development of parasite specific GS inhibitors for treatment of visceral leishmaniasis infections.

Characterization of plant glutamine synthetase S-nitrosation.

The identification of S-nitrosated substrates and their target cysteine residues is a crucial step to understand the signaling functions of nitric oxide (NO) inside the cells. Here, we show that the key nitrogen metabolic enzyme glutamine synthetase (GS) is a S-nitrosation target in Medicago truncatula and characterize the molecular determinants and the effects of this NO-induced modification on different GS isoenzymes. We found that all the four M. truncatula GS isoforms are S-nitrosated, but despite the high percentage of amino acid identity between the four proteins, S-nitrosation only affects the activity of the plastid-located enzymes, leading to inactivation. A biotin-switch/mass spectrometry approach revealed that cytosolic and plastid-located GSs share an S-nitrosation site at a conserved cysteine residue, but the plastidic enzymes contain additional S-nitrosation sites at non-conserved cysteines, which are accountable for enzyme inactivation. By site-directed mutagenesis, we identified Cys369 as the regulatory S-nitrosation site relevant for the catalytic function of the plastid-located GS and an analysis of the structural environment of the SNO-targeted cysteines in cytosolic and plastid-located isoenzymes explains their differential regulation by S-nitrosation and elucidates the mechanistic by which S-nitrosation of Cys369 leads to enzyme inactivation. We also provide evidence that both the cytosolic and plastid-located GSs are endogenously S-nitrosated in leaves and root nodules of M. truncatula, supporting a physiological meaning for S-nitrosation. Taken together, these results provide new insights into the molecular details of the differential regulation of individual GS isoenzymes by NO-derived molecules and open new paths to explore the biological significance of the NO-mediated regulation of this essential metabolic enzyme.

Molecular characterization of a novel algal glutamine synthetase (GS) and an algal glutamate synthase (GOGAT) from the colorful outer mantle of the giant clam, Tridacna squamosa, and the putative GS-GOGAT cycle in its symbiotic zooxanthellae.

Giant clams harbor symbiotic zooxanthellae (Symbiodinium), which are nitrogen-deficient, mainly in the fleshy and colorful outer mantle. This study aimed to sequence and characterize the algal Glutamine Synthetase (GS) and Glutamate Synthase (GLT), which constitute the glutamate synthase cycle (or GS-GOGAT cycle, whereby GOGAT is the protein acronym of GLT) of nitrogen assimilation, from the outer mantle of the fluted giant clam, Tridacna squamosa. We had identified a novel GS-like cDNA coding sequence of 2325 bp, and named it as T. squamosa Symbiodinium GS1 (TSSGS1). The deduced TSSGS1 sequence had 774 amino acids with a molecular mass of 85 kDa, and displayed the characteristics of GS1 and Nucleotide Diphosphate Kinase. The cDNA coding sequence of the algal GLT, named as T. squamosa Symbiodinium GLT (TSSGLT), comprised 6399 bp, encoding a protein of 2133 amino acids and 232.4 kDa. The zooxanthellal origin of TSSGS1 and TSSGOGAT was confirmed by sequence comparison and phylogenetic analyses. Indeed, TSSGS1 and TSSGOGAT were expressed predominately in the outer mantle, which contained the majority of the zooxanthellae. Immunofluorescence microscopy confirmed the expression of TSSGS1 and TSSGOGAT in the cytoplasm and the plastids, respectively, of the zooxanthellae in the outer mantle. It can be concluded that the symbiotic zooxanthellae of T. squamosa possesses a glutamate synthase (TSSGS1-TSSGOGAT) cycle that can assimilate endogenous ammonia produced by the host clam into glutamate, which can act as a substrate for amino acid syntheses. Thus, our results provide insights into why intact giant clam-zooxanthellae associations do not excrete ammonia under normal circumstances.

Characterization of an L-phosphinothricin resistant glutamine synthetase from Exiguobacterium sp. and its improvement.

A glutamine synthetase (GS; 1341 bp) gene with potent L-phosphinothricin (PPT) resistance was isolated and characterized from a marine bacterium Exiguobacterium sp. Molecular docking analysis indicated that the substitution of residues Glu60 and Arg64 may lead to significant changes in binding pocket. To enhance the enzymatic property of GS, variants E60A and R64G were obtained by site-directed mutagenesis. The results revealed a noteworthy change in the thermostability and activity in comparison to the wild type (WT). WT exhibited optimum activity at 35 °C, while E60A and R64G exhibited optimum activity at 45 and 40 °C, respectively. The mutant R64G was 4.3 times more stable at 70 °C in comparison to WT, while E60A was 5.7 times more stable. Kinetic analysis revealed that the k cat value of R64G mutant was 8.10-, 7.25- and 7.63-fold that of WT for ADP, glutamine and hydroxylamine, respectively. The kinetic inhibition (K i, 4.91 ± 0.42 mM) of R64G was 2.02-fold that of WT (2.43 ± 0.14 mM) for L-phosphinothricin. The analysis of structure and function relationship showed that the binding pocket underwent dramatic changes when Arg site of 64 was substituted by Gly, thus promoting the rapid capture of substrates and leading to increase in activity and PPT-resistance of mutant R64G. The rearrangements of the residues at the molecular level formed new hydrogen bonds around the active site, which contributed to the increase of thermostability of enzymes. This study provides new insights into substrate binding mechanism of glutamine synthetase and the improved GS gene also has a potential for application in transgenic crops with L-phosphinothricin tolerance.

First report on the occurrence of the uncultivated cluster 2 Frankia microsymbionts in soil outside the native actinorhizal host range area.

The occurrence of uncultivated Frankia was evaluated in Tunisian soils by a plant-trapping assay using Coriaria myrtifolia seedlings. Despite the lack of this compatible host plant for more than two centuries, soil-borne Frankia cells were detected in one sampled soil as shown by the development of root nodules on 2-year-old seedlings. Based on glnA sequences, Tunisian trapped Frankia strains belong to the uncultivated cluster 2 strains that associate with other Coriaria species and also with Ceanothus, Datisca and Rosaceae actinorhizal species. This is the first report on the occurrence of Frankia cluster 2 strains in soils from areas lacking compatible host plant groups.

Wanted and wanting: antibody against methionine sulfoxide.

Methionine residues in protein can be oxidized by reactive oxygen or nitrogen species to generate methionine sulfoxide. This covalent modification has been implicated in processes ranging from normal cell signaling to neurodegenerative diseases. A general method for detecting methionine sulfoxide in proteins would be of great value in studying these processes, but development of a chemical or immunochemical technique has been elusive. Recently, an antiserum raised against an oxidized corn protein, DZS18, was reported to be specific for methionine sulfoxide in proteins (Arch. Biochem. Biophys. 485:35-40; 2009). However, data included in that report indicate that the antiserum is not specific. Utilizing well-characterized native and methionine-oxidized glutamine synthetase and aprotinin, we confirm that the antiserum does not possess specificity for methionine sulfoxide.

A functional recombinant human 4-1BB ligand for immune costimulatory therapy of cancer.

Costimulatory factors hold great promise for development into novel anticancer biotherapeutics. An agonist to 4-1BB is ranked number 8 by National Cancer Institute on the list of 20 agents with high potential for use in treating cancer. We earlier reported on a recombinant murine 4-1BB ligand fusion protein that binds 4-1BB receptor on murine T cells and stimulates their proliferation in tumor-bearing mice. To facilitate clinical translation,we constructed a corresponding recombinant human 4-1BB ligand fusion protein (hIg-h4-1BBLs) and showed its ability to activate human T cells in vitro. Using Chinese hamster ovary cells transformed with a plasmid coexpressing hIg-h4-1BBLs and rat glutamine synthetase, we generated a high-producing clone by sequential selection with methionine sulfoximine. The hIg-h4-1BBLs was partially purified by protein A column chromatography and characterized biochemically and functionally, using human 4-1BB binding and human T-cell proliferation assays, in vitro.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western Blot confirmed that the hIg-h4-1BBLs is expressed predominantly as a functionally active multimeric protein with the ability to specifically bind to cells expressing human 4-1BB receptor and induce significant T-cell proliferation in vitro using both human and monkey peripheral blood mononuclear cells. The hIg-h4-1BBLs can be produced in large quantities from the high producer clone and developed as a novel immune costimulatory biotherapeutic to treat, alone and in combination with other modalities, various malignant diseases in patients through T-cell activation. Process development of this clinical agent has been discussed with the Food and Drug Administration in a pre-Investigational New Drug meeting and presented to the Office of Biotechnology Activities in a public hearing.

Crystallization of recombinant Bacteroides fragilis glutamine synthetase (GlnN) isolated using a novel and rapid purification protocol.

Glutamine synthetase enzymes (GSs) are large oligomeric enzymes that play a critical role in nitrogen metabolism in all forms of life. To date, no crystal structures exist for the family of large (∼1 MDa) type III GS enzymes, which only share 9% sequence identity with the well characterized GSI and GSII enzymes. Here we present a novel protocol for the isolation of untagged Bacteroides fragilis GlnN expressed in an auxotrophic Escherichia coli strain. The rapid and scalable two-step protocol utilized differential precipitation by divalent cations followed by affinity chromatography to produce suitable quantities of homogenous material for structural characterization. Subsequent optimizations to the sample stability and solubility led to the discovery of conditions for the production of the first diffraction quality crystals of a type III GS enzyme.

Effectiveness and mode of action of phosphonate inhibitors of plant glutamine synthetase.

BACKGROUND: Aiming at the rational design of new herbicides, the availability of the three-dimensional structure of the target enzyme greatly enhances the optimisation of lead compounds and the design of derivatives with increased activity. Among the most widely exploited herbicide targets is glutamine synthetase. Recently, the structure of a cytosolic form of the maize enzyme has been described, making it possible to verify whether steric, electronic and hydrophobic features of a compound are in agreement with inhibitor-protein interaction geometry. RESULTS: Three series of compounds (aminophosphonates, hydroxyphosphonates and aminomethylenebisphosphonates) were evaluated as possible inhibitors of maize glutamine synthetase. Aminomethylenebisphosphonate derivatives substituted in the phenyl ring retained the inhibitory potential, whereas variations in the scaffold, i.e. the replacement of the second phosphonate moiety with a hydroxyl or an amino residue, resulted in a significant loss of activity. A kinetic characterisation showed a non-competitive mechanism against glutamate and an uncompetitive mechanism against ATP. A docking analysis suggested the mode of bisphosphonate binding to the active site. CONCLUSION: Results made it possible to define the features required to maintain or enhance the biological activity of these compounds, which represent lead structures to be further exploited for the design of new substances endowed with herbicidal activity.

A biotin-coupled bifunctional enzyme exhibiting both glutamine synthetase activity and glutamate decarboxylase activity.

PURPOSE: To purify and study native form and enzymatic activity of the 42 kDa biotin-coupled protein (p42), which is related to glutamate action in chick retina. METHODS: p42 was purified using molecular filtration in the presence of 0.7 M sodium chloride. Purity and identification of p42 were studied by SDS-PAGE, 2D-PAGE, LC-MS/MS, and MALDI-TOF MS. The native form of p42 was investigated using native-PAGE and Ferguson plot. Biotin-coupled property was examined by Western blot analysis. Enzymatic actions of p42 were studied using glutamate as substrate in the presence or absence of glutamine. RESULTS: p42 was successfully purified from chick retinal protein solution using the molecular filtration. Western blot analysis with avidin showed that p42 was a biotin-coupled protein. Using SDS-PAGE, 2D-PAGE, LC-MS/MS, and MALDI-TOF MS, purified p42 was identified as a glutamine synthetase with four isoforms. Native-PAGE, followed by Ferguson plot analysis, showed two molecular forms of p42 corresponding to homotetramers and homooctamers. Enzymatic reaction followed by paper chromatography showed that p42 catalyzed the synthesis of glutamine from glutamate in the presence of ammonium ion, ATP, and magnesium ion. At prolonged reaction time, gamma-aminobutyric acid (GABA) was also formed. With glutamate and glutamine present at equal concentrations in the reaction mixture, GABA could be rapidly detected, but GABA could not be detected when glutamate concentration was more than four-fold that of glutamine. The results indicated that p42 also had glutamate decarboxylase activity. Both enzymatic activities were inhibited by avidin. High concentrations of Mn(2+) inhibited synthetase activity of p42 but not decarboxylase activity. CONCLUSION: p42 was purified from chick retinal protein solution using molecular filtration in the presence of sodium chloride. The protein was a biotin-coupled bifunctional enzyme that contained glutamine synthetase activity and glutamate decarboxylase activity. Biotin was possibly involved in these activities. Mn(2+) showed different effects on the two activities.

Candidate molecular markers for histological diagnosis of early hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. HCC occurs mainly in chronically diseased livers, e.g. following hepatitis B and C infection. These high-risk patients are closely followed up, and increasing numbers of small equivocal lesions are detected by imaging diagnosis. They are now widely recognized as precursor or early-stage HCCs and are classified as dysplastic nodule or early HCC. These lesions lack typical imaging and histology of ordinary HCC and do not show elevated serum markers of alpha-fetoprotein and PIVKA-II, for example. Molecular analysis of these lesions would help to develop molecular markers for objective histological diagnosis of early HCC and possibly new serum markers for early detection of HCC. It has been reported that HSP70, CAP2, glypican 3 and glutamine synthetase could serve as molecular markers for early HCC. Further analysis is expected to evaluate their usefulness in routine pathological diagnosis including biopsy diagnosis and also as serum markers for early detection of HCC.

Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design.

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 A. 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 A and 2.6 A, 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.

The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by alpha-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro.

Ammonium assimilation is tightly regulated in nitrogen-fixing bacteria; the target of regulation is primarily the activity of the key enzyme glutamine synthetase that is regulated by reversible covalent modification by AMP groups in reactions catalysed by the bifunctional adenylyltransferase (ATase). The properties and regulation of ATase from Escherichia coli have been studied in great detail. We have investigated the regulation of ATase from Rhodospirillum rubrum, a photosynthetic nitrogen-fixing bacterium. In this diazotroph, nitrogenase is regulated at the metabolic level in addition to the transcriptional regulation operating in all diazotrophic bacteria, which makes understanding the regulatory features of nitrogen assimilation even more interesting. We show that in R. rubrum, in contrast to the E. coli system, ATase is primarily regulated by alpha-ketoglutarate and that glutamine has no effect on neither the adenylylation nor the deadenylylation of glutamine synthetase. Furthermore, the role of the regulatory P(II) proteins is only to stimulate the adenylylation reaction, as there is no effect on the reverse reaction. We propose that in R. rubrum and possibly other diazotrophs alpha-ketoglutarate plays the central role in the regulation of ATase and thus glutamine synthetase activity.

Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production.

The roles of two cytosolic maize glutamine synthetase isoenzymes (GS1), products of the Gln1-3 and Gln1-4 genes, were investigated by examining the impact of knockout mutations on kernel yield. In the gln1-3 and gln1-4 single mutants and the gln1-3 gln1-4 double mutant, GS mRNA expression was impaired, resulting in reduced GS1 protein and activity. The gln1-4 phenotype displayed reduced kernel size and gln1-3 reduced kernel number, with both phenotypes displayed in gln1-3 gln1-4. However, at maturity, shoot biomass production was not modified in either the single mutants or double mutants, suggesting a specific impact on grain production in both mutants. Asn increased in the leaves of the mutants during grain filling, indicating that it probably accumulates to circumvent ammonium buildup resulting from lower GS1 activity. Phloem sap analysis revealed that unlike Gln, Asn is not efficiently transported to developing kernels, apparently causing reduced kernel production. When Gln1-3 was overexpressed constitutively in leaves, kernel number increased by 30%, providing further evidence that GS1-3 plays a major role in kernel yield. Cytoimmunochemistry and in situ hybridization revealed that GS1-3 is present in mesophyll cells, whereas GS1-4 is specifically localized in the bundle sheath cells. The two GS1 isoenzymes play nonredundant roles with respect to their tissue-specific localization.

Purification and properties of glutamine synthetase from Hydrogenobacter thermophilus TK-6.

Hydrogenobacter thermophilus TK-6, a thermophilic and obligately chemoautotrophic bacterium, assimilates ammonium using glutamine synthetase (GS). GS was purified using three chromatography steps. The purified GS was found to belong to GS type I on the basis of its subunit composition and molecular weight. The Mg2+ -dependent activity of this GS significantly increased after incubation with phosphodiesterase, indicating that GS is subject to adenylyl/deadenylyl regulation, a posttranslational modification system reported mainly among enterobacteria. The degree of this posttranslational modification changed depending on growth phase, confirming that adenylyl/deadenylyl regulation functions in vivo. Interestingly, the Km for glutamate of H. thermophilus GS was significantly higher than those of other organisms, suggesting that GS activity is affected by intracellular glutamate concentration.

An octameric prokaryotic glutamine synthetase from the haloarchaeon Haloferax mediterranei.

The glutamine synthetase (EC 6.3.1.2) from the haloarchaeon Haloferax mediterranei has been purified and characterized in order to understand the ammonium assimilation in haloarchaea. Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel-filtration chromatography, the enzyme consists of eight subunits of 51.7 kDa, suggesting that this enzyme belongs to the glutamine synthetase type II. The purified enzyme has been characterized with respect to its optimum temperature (45 degrees C) and pH value (8.0). The optimal NaCl or KCl concentrations for the reaction were 0.5 and 0.25 M, respectively. The effect of l-methionine-d, l-sulphoximine and different divalent metal ions has also been tested. The glutamine synthetase presented here is unusual; it shows the typical characteristic of eukaryotic and soil bacteria glutamine synthetases.

Three-dimensional structure of a type III glutamine synthetase by single-particle reconstruction.

GlnN, the type III glutamine synthetase (GSIII) from the medically important, anaerobic, opportunistic pathogen Bacteroides fragilis, has 82.8 kDa subunits that share only 9% sequence identity with the type I glutamine synthetases (GSI), the only family for which a structure is known. Active GlnN was found predominantly in a single peak that eluted from a calibrated gel-filtration chromatography column at a position equaivalent to 0.86(+/-0.08) MDa. Negative-stain electron microscopy enabled the identification of double-ringed particles and single hexameric rings ("pinwheels") resulting from partial staining. A 2D average of these pinwheels showed marked similarity to the corresponding structures found in preparations of GSI, except that the arms of the subunits were 40% longer. Reconstructions from particles embedded in vitreous ice showed that GlnN has a double-ringed, dodecameric structure with a 6-fold dihedral space group (D6) symmetry and dimensions of 17.0 nm parallel with the 6-fold axis and 18.3 nm parallel with the 2-fold axes. The structures, combined with a sequence alignment based on structural principles, showed how many aspects of the structure of GSI, and most notably the alpha/beta barrel fold active site were preserved. There was evidence for the presence of this structure in the reconstructed volume, thus, identifying the indentations between the pinwheel spokes as putative active sites and suggesting conservation of the overall molecular geometry found in GSI despite their low level of global homology. Furthermore, docking of GSI into the reconstruction left sufficient plausibly located unoccupied density to account for the additional residues in GSIII, thus validating the structure.

Molecular analysis of two mutants from Lotus japonicus deficient in plastidic glutamine synthetase: functional properties of purified GLN2 enzymes.

Two photorespiratory mutants from Lotus japonicus, namely Ljgln2-1 and Ljgln2-2, deficient in plastidic glutamine synthetase (GLN2), were analysed at the molecular level. Both mutants showed normal levels of Gln2 mRNA, indicating that they were affected post-transcriptionally. Complete sequencing of full-length Gln2 cDNAs revealed the presence of a single point mutation on each mutant, leading to G85R and L278H amino acid replacements, respectively. Different types of experimental approaches, including heterologous expression and complementation tests in Escherichia coli, showed that both GLN2 mutant proteins completely lacked of biosynthetic and transferase enzyme activities. Moreover, it was also shown that while GLN2-1 mutant protein was assembled into a less stable inactive octamer, GLN2-2 mutant protein was unable to acquire a proper quaternary structure and was rapidly degraded. Therefore, the mutations analysed are the first of their type affecting the stability and/or the quaternary structure of the GLN2 enzyme. The kinetic parameters of purified recombinant GLN2 were determined. The enzyme showed positive cooperativity towards ammonium and Mg(2+). Thiol compounds stimulated by twofold the biosynthetic activity but not the transferase activity of recombinant GLN2 and were able to alter the kinetics towards glutamate of the enzyme. Moreover, the biosynthetic activity of recombinant GLN2 was stimulated by more than tenfold by the presence of free Mg(2+).

Phosphinothricin analogues as inhibitors of plant glutamine synthetases.

A series of phosphinothricin derivatives with a modified methyl group, designed on the basis of the crystal structure of the complex formed by the inhibitor and the target enzyme from Salmonella typhimurium, were evaluated as potential inhibitors of plant glutamine synthetase. These compounds were previously shown to be equipotent or slightly weaker inhibitors to the lead compound against the bacterial enzyme. Because of the presence in higher plants of at least two enzyme forms with different subcellular localization and possible separate metabolic functions, plastidial and cytosolic glutamine synthetases were purified to electrophoretic homogeneity from spinach chloroplasts and cultured tobacco cells, respectively. Kinetic analysis confirmed the ability of the phosphinothricin analogues to inhibit both isoenzymes in the micromolar range, with a mechanism of a competitive type with respect to glutamate. Interestingly, some of them exerted a differential effect against either the two plant isoforms, or against the plant versus the bacterial enzyme.

Cloning and expression of Pseudomonas taetrolens Y-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.