Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6?kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53?269?Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205?kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80°?C; ammonia-dependent reaction, 90°?C).     

Root and Nodule Enzymes of Ammonia Assimilation in Two Plant-Conditioned Symbiotically Ineffective Genotypes of Alfalfa (Medicago sativa L.)

Biochemical and physiological parameters associated with nitrogen metabolism were measured in nodules and roots of glasshouse-grown clones of two symbiotically ineffective alfalfa (Medicago sativa L.) genotypes supplied with either NO₃⁻ or NH₄⁺. Significant differences were observed between genotypes for nodule soluble protein concentrations and glutamine synthetase (GS) and glutamate synthase (GOGAT) specific activities, both in untreated controls and in response to applied N. Nodule soluble protein of both genotypes declined in response to applied N, while nodule GS, GOGAT, and glutamate dehydrogenase (GDH) specific activities either decreased or remained relatively constant. In contrast, no genotype differences were observed in roots for soluble protein concentrations and GS, GOGAT, and GDH specific activities, either in untreated controls or in response to applied N. Root soluble protein levels and GS and GOGAT specific activities of N-treated plants increased 2- to 4-fold within 4 days and then decreased between days 13 and 24. Root GDH specific activity of NH₄⁺-treated plants increased steadily throughout the experiment and was 50 times greater than root GS or GOGAT specific activities by day 24.     

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N₂-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N₂-fixing) alfalfa recessive for the in₁ gene were compared to determine the effects of the in₁ gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in₁ effective nodules.     

Expression and functional analysis of glutamate synthase small subunit-like proteins from archaeon Pyrococcus horikoshii

Glutamate synthase, glutamine α-ketoglutarate amidotransferase (often abbreviated as GOGAT) is a key enzyme in the early stages of ammonia assimilation in bacteria, algae and plants, catalyzing the reductive transamidation of the amido nitrogen from glutamine to α-ketoglutarate to form two molecules of glutamate. Most bacterial glutamate synthases consist of a large and small subunit. The genomes of three Pyrococcus species harbour several open reading frames which show homology with the small subunit of glutamate synthase. There are no open reading frames which may be coding for a large subunit responsible for the glutamate formation in these pyrococcal genomes.In this work, two open reading frames PH0876 and PH1873 from P. horikoshii were cloned and expressed in Escherichia coli as soluble proteins. Both proteins show NADPH-dependent oxidoreductase activity using artificial electron acceptors iodonitrotetrazolium chloride at thermophilic conditions. It is possible that these open reading frames are the products of gene duplication and that they are the early forms of an electron transfer domain in archaea which may have later contributed to many electron transfer enzymes.     

Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.     

Regulation and function of glutamate synthase in Neurospora crassa

In Neurospora crassa two enzymes can provide glutamate: the NADPH dependent GDH and the NADH dependent GOGAT. An elevated GOGAT activity was found in Neurospora wild-type under ammonium limitation in contrast to a 4-fold lower activity on excess of a$\text{m}$ monium. Glutamate and glutamine repress this enzyme. On excess of ammonium the GDH-NADPH deficient mutant am-1 grows poorly with an elevated GOGAT activity. A GOGAT less mutant was found. It presented a lag-phase to grow on ammonium. It is concluded that N. crassa glutamate synthase provides glutamate from low am-monium concentrations. The enzyme was purified to homogeneity and shown to be composed of a single type of monomer with a molecular weight above 200,000.     

Glutamate formation by a new In vitro enzyme system consisting of purified glutamine synthetase and glutamate synthase

After the addition of ammonia to the culture medium, the concentration of glutamine in B. flavum cells increased in 20 s with a decrease in glutamate. In the subsequent 30 s, the glutamine concentration deceased again with an increase in glutamate. An enzyme system, which consisted of purified glutamine synthetase (GS) and glutamate synthase (GOGAT) with ATP- and NADPH-regenerating systems, was made up to study the functions of the GS/GOGAT pathway: concentrations of the substrates and of the enzymes were decided on according to the intracellular conditions. Changes in the concentrations of amino acids caused by the addition of ammonia to the system were very similar to those of intracellular glutamate and glutamine when ammonia was added to the bacterial culture. The time required for the complete formation of glutamate from 0.5 mM ammonia was about 4-times shorter in the GS/GOGAT system than in the system using purified glutamate dehydrogenase (GDH) and the NADPH-regenerating system. The glutamate synthase reaction in the GS/GOGAT system was inhibited by some amino acids much more markedly than in the standard assay mixture consisting of glutamine, α-ketoglutarate and NADPH. These results gave further evidence elucidating the operation of the GS/GOGAT pathway in ammonia assimilation, and suggested that a reconstructed enzyme system is useful for studying physiological mechanisms.     

Synthesis of glutamine and glutamate by intact bundle-sheath cells of maize (Zea mays L.)

Intact bundle-sheath cells with functional plasmodesmata were isolated from leaves of Zea mays L. cv. Mutin, and the capacity of these cells to synthesize glutamine and glutamate was determined by simulating physiological substrate concentrations in the bathing medium. The results show that glutamine synthetase can operate at full rate in the presence of added 8 mM ATP. At lower concentrations of ATP a higher rate of glutamine synthesis was found in the light than in darkness. Glutamate-synthase activity, on the other hand, was strictly light dependent. It appears that in bundle-sheath cells of maize the nitrate-assimilatory capacities of glutamine synthetase (located mainly in the cytosol) and of glutamate synthase (located in the stroma) are high enough to meet the demands of whole maize leaves.Abbreviations Gln glutamine - Glu glutamate - GOGAT glutamate synthase - GS glutamine synthetase - 2-OG 2-oxoglutarate This work was supported by the Bundesminister für Forschung und Technologie (0319296A). We thank Mr. Bernd Raufeisen for the art work of Fig. 1.     

Cellular localization of NADH-dependent glutamate-synthase protein in vascular bundles of unexpanded leaf blades and young grains of rice plants

Tissue and cellular localization of NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) in the unexpanced leaf blades and young grains of rice (Oryza sativa L.) was investigated using tissue-print immunoblot and immunocytological methods with an affinity-purified anti-NADH-GOGAT immunoglobulin G. Tissue-print immunoblots showed that the NADH-GOGAT protein was mostly located in large and small vascular bundles of the unexpanded blades. When the cross-sections (10μ in thickness) prepared from the paraffin-embedded blades were stained with the antibody, the NADH-GOGAT protein was detected in vascular-parenchyma cells and mestome-sheath cells. In developing grains, the NADH-GOGAT protein was detected in both phloem- and xylem-parenchyma cells of dorsal and lateral vascular bundles, and in the nucellar projection, nucellar epidermis, and aleurone cells. On the other hand, ferredoxin (Fd)-dependent GOGAT (EC 1.4.7.1) was located mainly in mesophyll cells of the leaf blade and in chloroplast-containing cross-cells of the pericarp of the grains. The spatial expression of these GOGAT proteins indicates distinct and non-overlapping roles in rice plants. In the leaf blades and young grains, NADH-GOGAT could be involved in the synthesis of glutamate from the glutamine that is transported through the vascular system from roots and senescing tissues.     

Glutamine synthetase and glutamate synthase from Sclerotinia sclerotiorum

Glutamine synthetase (GS, EC 6.3.1.2) and glutamate synthase (GOGAT, EC 1.4.1.13) were purified from Sclerotinia sclerotiorum and some of their properties studied. The GS transferase and biosynthetic activities, as well as GOGAT activity, were sensitive to feedback inhibition by amino acids and other metabolites. GS showed a marked dependence on ADP in the transferase reaction and on ATP in the Mg²⁺-dependent biosynthetic reaction. Regulation of GS activity by adenylylation/deadenylylation was demonstrated by snake venom phosphodiesterase treatment of the purified enzyme. GOGAT required NADPH as an electron donor; NADH was inactive. GOGAT was strongly inhibited by p-chloromercuribenzoate and the inhibition was reversed by cysteine. The enzyme was also markedly inhibited by o-phenanthroline, 2,2′-bipyridyl and azaserine. l-Methionine-dl-sulphoximine (MSX) and azaserine inhibited the incorporation of ¹⁵N-labelled ammonium sulphate into washed cells of S. sclerotiorum. MSX and azaserine respectively also inhibited purified GS and GOGAT activities. GDH activity was not detected in cell-extracts. Thus the GS/GOGAT pathway is the main route for the assimilation of ammonium compounds in this fungus.     

Purification and Characterization of NADH-Glutamate Synthase from Alfalfa Root Nodules

Glutamate synthase (GOGAT), a key enzyme in the pathway for the assimilation of symbiotically fixed dinitrogen (N₂) into amino acids in alfalfa (Medicago sativa L.) root nodules, was purified and used to produce high titer polyclonal antibodies. Purification resulted in a 208-fold increase in specific activity to 13 micromole per minute per milligram of protein and an activity yield of 37%. Further purification to near homogeneity was achieved by fast protein liquid chromatography, but with substantial loss of activity. Enzymic activity was highly labile, losing 3% per hour even when substrates, stabilizers, and reducing agents were included in buffers. However, activity could be partially stabilized for up to 1 month by storing GOGAT at −80°C in 50% glycerol. The subunit molecular weight of GOGAT was estimated at 200 ± 7 kilodaltons with a native molecular weight of 235 ± 16 kilodaltons, which suggested that GOGAT is a monomer of unusually high molecular weight. The pl was estimated to be 6.6. The K_m values for glutamine, α-ketoglutarate, and NADH were 466, 33, and 4.2 micromolar, respectively. Antibodies were produced to NADH-GOGAT. Specificity of the antibodies was shown by immunotitration of GOGAT activity. Alfalfa nodule NADH-GOGAT antibodies cross-reacted with polypeptides of a similar molecular weight in a number of legume species. Western blots probed with anti-GOGAT showed that the high GOGAT activity of nodules as compared to roots was associated with increased levels of GOGAT polypeptides. Nodule NADH-GOGAT appeared to be highly expressed in effective nodules and little if any in other organs.     

Enzymes of ammonium assimilation inStreptomyces avermitilis

Glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), alanine dehydrogenase (ADH) and alanine aminotransferase (GPT) were detected in the cell-free homogenate ofStreptomyces avermitilis grown in a defined medium containing ammonium sulfate as the only nitrogen source. At an initial NH₄ ⁺ concentration of 7.5 mmol/L, high activities of GS, GOGAT and GDH were found while that of ADH was low. The ADH activity was markedly increased at initially millimolar NH₄ ⁺ concentrations. In some characteristics of its NH₄ ⁺-assimilating system (e.g. control of some enzyme activities, the NADPH specificity of GOGAT, the presence of alanine aminotransferase),S. avermitilis differs from other known streptomycetes.     

Oxidation of the enzymes involved in nitrogen assimilation plays an important role in the cadmium-induced toxicity in soybean plants

Cadmium causes oxidative damage and hence affects nitrogen assimilation. In the present work we tested the relationship between the inactivation of the enzymes involved in nitrogen assimilation pathway (glutamine synthetase (GS)/glutamate synthase (GOGAT)) and the protein oxidation in nodules of soybean (Glycine max L.) plants under Cd²⁺ stress. Therefore, the effect of Cd²⁺ and reduced gluthatione (GSH) on GS and GOGAT activities, and protein abundance and oxidation were analyzed. Under the metal treatment, amino acids oxidative modification occurred, evidenced by the accumulation of carbonylated proteins, especially those of high molecular weight. When Cd²⁺ was present in the nutrient solution, although a decrease in GS and GOGAT activities was observed (17 and 52%, respectively, compared to controls), the protein abundance of both enzymes remained similar to control nodules. When GSH was added together with Cd²⁺ in the nutrient medium, it protected the nodule against Cd²⁺ induced oxidative damage, maintaining GS and GOGAT activities close to control values. These results allow us to conclude that the inactivation of the nitrogen assimilation pathway by Cd²⁺ in soybean nodules is due to an increment in GS and GOGAT oxidation that can be prevented by the soluble antioxidant GSH. Section Editor: H. Schat     

The Role of Glutamine Oxoglutarate Aminotransferase and Glutamate Dehydrogenase in Nitrogen Metabolism in Mycobacterium bovis BCG

Recent evidence suggests that the regulation of intracellular glutamate levels could play an important role in the ability of pathogenic slow-growing mycobacteria to grow in vivo. However, little is known about the in vitro requirement for the enzymes which catalyse glutamate production and degradation in the slow-growing mycobacteria, namely; glutamine oxoglutarate aminotransferase (GOGAT) and glutamate dehydrogenase (GDH), respectively. We report that allelic replacement of the Mycobacterium bovis BCG gltBD-operon encoding for the large (gltB) and small (gltD) subunits of GOGAT with a hygromycin resistance cassette resulted in glutamate auxotrophy and that deletion of the GDH encoding-gene (gdh) led to a marked growth deficiency in the presence of L-glutamate as a sole nitrogen source as well as reduction in growth when cultured in an excess of L-asparagine.     

The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent K _m values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent K _m values were 0.1 mM for -ketoglutarate and 0.22 mM for glutamine.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase     

Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53 269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80° C; ammonia-dependent reaction, 90° C). Received: 30 October 1996 / Accepted: 13 January 1997     

NADH‐dependent glutamate synthase plays a crucial role in assimilating ammonium in the Arabidopsis root

Plant roots under nitrogen deficient conditions with access to both ammonium and nitrate ions, will take up ammonium first. This preference for ammonium rather than nitrate emphasizes the importance of ammonium assimilation machinery in roots. Glutamine synthetase (GS) and glutamate synthase (GOGAT) catalyze the conversion of ammonium and 2‐oxoglutarate to glutamine and glutamate. Higher plants have two GOGAT species, ferredoxin‐dependent glutamate synthase (Fd‐GOGAT) and nicotinamide adenine dinucleotide (NADH)‐GOGAT. While Fd‐GOGAT participates in the assimilation of ammonium, which is derived from photorespiration in leaves, NADH‐GOGAT is highly expressed in roots and its importance needs to be elucidated. While ammonium as a minor nitrogen form in most soils is directly taken up, nitrate as the major nitrogen source needs to be converted to ammonium prior to uptake. The aim of this study was to investigate and quantify the contribution of NADH‐GOGAT to the ammonium assimilation in Arabidopsis (Arabidopsis thaliana Columbia) roots. Quantitative real‐time polymerase chain reaction (PCR) and protein gel blot analysis showed an accumulation of NADH‐GOGAT in response to ammonium supplied to the roots. In addition the localization of NADH‐GOGAT and Fd‐GOGAT did not fully overlap. Promoter–β‐glucuronidase (GUS) fusion analysis and immunohistochemistry showed that NADH‐GOGAT was highly accumulated in non‐green tissue like vascular bundles, shoot apical meristem, pollen, stigma and roots. Reverse genetic approaches suggested a reduction in glutamate production and biomass accumulation in NADH‐GOGAT transfer DNA (T‐DNA) insertion lines under normal CO₂ condition. The data emphasize the importance of NADH‐GOGAT in the ammonium assimilation in Arabidopsis roots.     

15N and inhibitor studies on the photorespiratory nitrogen cycle in maize leaves

A combination of inhibitor and ¹⁵N studies were used to investigate the photorespiratory nitrogen cycle in maize, a C₄ plant. Inhibitors used included isonicotinyl hydrazide which blocks the conversion of glycine to serine, methionine sulfoximine an inhibitor of GS and azaserine an inhibitor of GOGAT. Results from levels of ammonia and amino acids and the distribution of ¹⁵N into NH₃, serine, glutamine and glutamate indicated that the photorespiratory N-cycle occurs in this C₄ plant, but the rate of flux through this pathway is low as compared with that in C₃ plants.Abbreviations Aza azasering - fw fresh weight - GOGAT glutamate synthase - GS glutamine synthetase - INH isonicotinyl hydrazide - MSO methionine sulfoximine     

Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm

Kernels of wild-type maize (Zea mays L.) shrunken-1 (sh1), deficient in the predominant form of endosperm sucrose synthase and shrunken-2 (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphorylase were grown in culture on sucrose, glucose, or fructose as the carbon source. Analysis of the endosperm extracts by gas-liquid chromatography revealed that sucrose was present in the endosperms of all genotypes, regardless of carbon supply, indicating that all three genotypes are capable of synthesizing sucrose from reducing sugars. The finding that sucrose was present in sh1 kernels grown on reducing sugars is evidence that shrunken-1 encoded sucrose synthase is not necessary for sucrose synthesis. Shrunken-1 kernels developed to maturity and produced viable seeds on all carbon sources, but unlike wild-type and sh2 kernels grown in vitro, sucrose was not the superior carbon source. This latter result provides further evidence that the role of sucrose synthase in maize endosperm is primarily that of sucrose degradation.     

Identification of granule-bound starch synthase in potato tubers

Starch granules isolated from potato (Solanum tuberosum L.) tubers were extracted with sodium dodecyl sulfate and the extract was analyzed. A major protein with a molecular weight of 60,000 daltons was detected. This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis and specific antibodies were prepared. The anti-60-kilodalton antibodies obtained (a) cross-reacted with the waxy proteins of both maize (Zea mays L.) and grain amaranth (Amaranthus hypochondriacus L.), and (b) inhibited starch synthase activity in partially digested starch granules of the grain amaranth. This evidence strongly suggests that the major 60-kilodalton protein present in potato starch granules represents the granule-bound starch synthase.