The enzymes of the ammonia assimilation in Pseudomonas aeruginosa

Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen while synthesis of the enzyme was repressed and that present was adenylylated in cultures with excess nitrogen.NADP-and NAD-dependent glutamate dehydrogenase could be separated by column chromatography and showed molecular weights of 110,000 and 220,000, respectively. Synthesis of the NADP-dependent glutamate dehydrogenase is repressed under nitrogen limitation and by growth on glutamate. In contrast, NAD-dependent glutamate dehydrogenase is derepressed by glutamate. Glutamate synthase is repressed by glutamate but not by excess nitrogen.     

Ammonia assimilation and glutamate formation in Caulobacter crescentus.

In the dimorphic bacterium Caulobacter crescentus, ammonia assimilation occurs only via the combined action of the enzymes glutamine synthetase and glutamate synthase. Mutants auxotrophic for glutamate lacked glutamate synthase activity, and the mutations leading to the glutamate auxotrophy appeared to lie at two distinct genetic loci. Both glutamate synthase and glutamine synthetase activities were subject to regulation by repression. Glutamate synthase activity was highest in cultures grown in minimal medium with ammonia as sole nitrogen source and was about fivefold lower in rich broth. Glutamine synthetase activity was highest in cells grown with growth-rate-limiting amounts of ammonia as nitrogen source and was about fourfold lower in rich broth. In addition, glutamine synthetase activity appeared to be regulated by an adenylylation system like that described for Escherichia coli.     

Ammonia assimilation by rhizobium cultures and bacteroids.

The enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.o.I.2), glutamate synthase (L-glutamine:2-oxoglutarate amino transferase) and glutamate dehydrogenase (ED I.4.I.4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slow-growing R. japonicum and R. lupini. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M. sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system.     

Nitrate reductase and glutamine synthetase activities,nitrate and ammonia assimilation,in the unicellular alga Cyanidium caldarium

Nitrogen-limited continuous cultures of Cyanidium caldarium contained induced levels of glutamine synthetase and nitrate reductase when either nitrate or ammonia was the sole nitrogen source. Nitrate reductase occurred in a catalytically active form. In the presence of excess ammonia, glutamine synthetase and nitrate reductase were repressed, the latter enzyme completely. In the presence of excess nitrate, intermediate levels of glutamine synthetase activity occurred. Nitrate reductase was derepressed but occurred up to 60% in a catalytically inactive form.Cell suspensions of C. caldarium from nitrate- or ammonialimited cultures assimilated either ammonia or nitrate immediately when provided with these nutrients. In these types of cells, as well as in cells grown with excess nitrate, the rate of ammonia assimilation was 2.5-fold higher than the rate of nitrate assimilation. It is proposed that the reduced rate at which nitrate was assimilated as compared to ammonia might be due to regulatory mechanisms which operate at the level of nitrate reductase activity.     

The anaerobic fungusPiromyces sp. strain E2: nitrogen requirement and enzymes involved in primary nitrogen metabolism

The anaerobic fungusPiromyces sp. strain E2 appeared restricted in nitrogen utilization. Growth was only supported by ammonium as source of nitrogen. Glutamine also resulted in growth, but this was due to release of ammonia rather than to uptake and utilization of the amino acid. The fungus was not able to grow on other amino acids, albumin, urea, allantoin, or nitrate. Assimilation of ammonium is very likely to be mediated by NADP-linked glutamate dehydrogenase (NADP-GDH) and glutamine synthetase (GS). One transaminating activity, glutamate-oxaloacetate transaminase (GOT), was demonstrated. Glutamate synthase (GOGAT), NAD-dependent glutamate dehydrogenase (NAD-GDH), and the transaminating activity glutamate-pyruvate transaminase (GPT) were not detected in cell-free extracts ofPiromyces sp. strain E2. Specific enzyme activities of both NADP-GDH and GS increased four-to sixfold under nitrogen-limiting conditions.Abbreviations GDH Glutamate dehydrogenase - GOGAT Glutamate synthase - GOT Glutamate-oxaloacetate transaminase - GPT Glutamate-pyruvate transaminase - GS Glutamine synthetase     

The role of ammonia, L-glutamate, and cyclic adenosine 3',5'-monophosphate in the regulation of ammonia assimilation in Rhizobium japonicum.

The effects of three factors (ammonia, L-glutamate, and cyclic adenosine 3',5'-monophosphate) on the ammonia assimilatory processes in aerobically grown Rhizobium japonicum colony derivatives were examined. Ammonia repressed glutamine synthetase activity and increased the average state of adenylylation of this enzyme. The addition of L-glutamate drastically decreased growth and strongly repressed glutamate synthase levels. Glutamine synthetase repression and adenylylation state were also increased by L-glutamate. The presence of cyclic AMP led to the repression of all three NH+4 assimilatory enzymes.     

The regulation of ammonia assimilating enzymes in Lemma minor

Summary Lemna minor has the potential to assimilate ammonia via either the glutamine or glutamate pathways. A 3-4 fold variation in the level of ferredoxindependent glutamate synthase may occur, when plants are grown on different nitrogen sources, but these changes show no simple relationship to changes in the endogenous pool of glutamate. High activities of glutamate synthase and glutamine synthetase at low ammonia availability suggests that these two enzymes function in the assimilation of low ammonia concentrations. Increasing ammonia availability leads to a reduction in level of glutamate synthase and glutamine synthetase and an increase in the level of glutamate dehydrogenase. Glutamine synthetase and glutamate dehydrogenase are subject to concurrent regulation, with glutamine rather than ammonia, exerting negative control on glutamine synthetase and positive control on glutamate dehydrogenase. The changes in the ratio of these two enzymes in response to the internal pool of glutamine could regulate the direction of the flow of ammonia into amino acids via the two alternative routes of assimilation.Abbreviations GS Glutamine synthetase - GDH Glutamate dehydrogenase - GOGAT Glutamate synthase     

The role of ammonia,L-glutamate,and cyclic adenosine 3′,5′-monophosphate in the regulation of ammonia assimilation in Rhizobium japonicum

The effects of three factors (ammonia, L-glutamate, and cyclic adenosine 3′,5′-monophosphate) on the ammonia assimilatory processes in aerobically grown Rhizobium japonicum colony derivatives were examined. Ammonia repressed glutamine synthetase activity and increased the average state of adenylylation of this enzyme. The addition of L-glutamate drastically decreased growth and strongly repressed glutamate synthase levels. Glutamine synthetase repression and adenylylation state were also increased by L-glutamate. The presence of cyclic AMP led to the repression of all three NH₄⁺ assimilatory enzymes.     

Glutamine- and Asparagine-Dependent Protein Synthesis in Maturing Legume Cotyledons Cultured in vitro

Legume cotyledons have been grown in sterile culture and shownto synthesize up to 16 mg protein in 5 d when supplied witheither asparagine or glutamine as sole nitrogen sources. Glutamate,nitrate, and ureides also served as nitrogen sources for proteinsynthesis but to a lesser extent. Methionine sulphoximine and azaserine inhibited asparagine-dependentprotein synthesis suggesting that ammonia is liberated fromasparagine which is reassimilated via glutamine synthetase andglutamate synthase.     

Evidence for a role of glutamine synthetase in assimilation of amino acids as nitrogen source in the cyanobacterium Nostoc muscorum.

Methylammonium/ammonium ion, glutamine, glutamate, arginine and proline uptake, and their assimilation as nitrogen sources, was studied in Nostoc muscorum and its glutamine synthetase-deficient mutant. Glutamine served as nitrogen source independent of glutamine synthetase activity. Glutamate was not metabolised as a nitrogen source but still inhibited nitrogenase activity and diazotrophic growth. Glutamine synthetase activity was essential for the assimilation of N2, ammonia, arginine and proline as nitrogen sources but not for the control of their transport, heterocyst formation, and production of ammonia or aminoacid dependent repressor signal for N2-fixing heterocysts. These results also suggest that glutamine synthetase serves as the sole route of ammonia assimilation and glutamine synthesis, and ammonia per se as the repressor signal for N2-fixing heterocysts and methylammonium (ammonium) transport.     

Studies onAspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grownAspergillus niger was increased 3–5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH ₄ ⁺ , and further, the enzyme is repressed by increasing concentrations of NH ₄ ⁺ . In contrast to other micro-organisms, theAspergillus niger enzyme was neither specifically inactivated by NH ₄ ⁺ or L-glutamine nor regulated by covalent modification. Glutamine synthetase fromAspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn²⁺ or Mg²⁺-dependent synthetase and Mn²⁺-dependent transferase activity. Aspergillusniger glutamine synthetase was completely inactivated by two mol of phenyl-glyoxal and one mol of N-ethylmaleimide with second order rate constants of 3.8 M^-1 min^-1 and 760 M^-1 min^-1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH ₄ ⁺ , Mn²⁺ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.     

Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium.

Urease and glutamine synthetase activities in Selenomonas ruminantium strain D were highest in cells grown in ammonia-limited, linear-growth cultures or when certain compounds other than ammonia served as the nitrogen source and limited the growth rate in batch cultures. Glutamate dehydrogenase activity was highest during glucose (energy)-limited growth or when ammonia was not growth limiting. A positive correlation (R = 0.96) between glutamine synthetase and urease activities was observed for a variety of growth conditions, and both enzyme activities were simultaneously repressed when excess ammonia was added to ammonia-limited, linear-growth cultures. The glutamate analog methionine sulfoximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamate dehydrogenase, glutamate synthase, and urease activities were not affected. The addition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammonia resulted in growth rate inhibition that was dependent upon the concentration of MSX and was overcome by glutamine addition. Urease activity in MSX-inhibited cultures was increased significantly, suggesting that ammonia was not the direct repressor of urease activity. In ammonia-limited, linear-growth cultures, MSX addition resulted in growth inhibition, a decrease in GS activity, and an increase in urease activity. These results are discussed with respect to the importance of glutamine synthetase and glutamate dehydrogenase for ammonia assimilation under different growth conditions and the relationship of these enzymes to urease.     

Regulation of the ammonia assimilatory enzymes in Salmonella typhimurium.

The regulation of glutamate dehydrogenase (EC 1.4.1.4), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 2.6.1.53) was examined for cultures of Salmonella typhimurium grown with various nitrogen and amino acid sources. In contrast to the regulatory pattern observed in Klebsiella aerogenes, the glutamate dehydrogenase levels of S. typhimurium do not decrease when glutamine synthetase is derepressed during growth with limiting ammonia. Thus, it appears that the S. typhimurium glutamine synthetase does not regulate the synthesis of glutamate dehydrogenase as reported for K. aerogenes. The glutamate dehydrogenase activity does increase, however, during growth of a glutamate auxotroph with glutamate as a limiting amino acid source. The regulation of glutamate synthase levels is complex with the enzyme activity decreasing during growth with glutamate as a nitrogen source, and during growth of auxotrophs with either glutamine or glutamate as limiting amino acids.     

Growth of Bacillus fastidiosus on glycerol and the enzymes of ammonia assimilation

Bacillus fastidiosus was able to grow on glycerol as a carbon source when allantoin or urate was used as nitrogen source. The primary assimilatory enzyme for glycerol was glycerol kinase; glycerol dehydrogenase could not be detected. The glycerol kinase activity was increased 30-fold in allantoin/glycerol-grown cells as compared to alantoin-grown cells. Under both growth conditions high levels of glutamate dehydrogenase were found. Glutamine synthetase and glutamate synthase activities could not be demonstrated, while low levels of alanine dehydrogenase were present. It is concluded that B. fastidiosus assimilates ammonia by the NADP-dependent glutamate dehydrogenase.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase     

Photoproduction of ammonium ion from N2 in Rhodospirillum rubrum.

NH+4 excretion was undetectable in N2-fixing cultures of Rhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH+4 to the medium. The glutamate analog, L-methionine-DL-sulfoximine (MSX), derepressed N2 fixation even in the presence of 10 mM extracellular NH+4. When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N2 as NH+4. Nitrogenase activities and NH+4 production from fixed N2 were increased considerably when a combined nitrogen source, NH+4 (greater than 40 mumoles NH+4/mg cell protein in 6 days) or L-glutamate (greater than 60 mumoles NH+4/ mg cell protein in 6 days) was added to the cultures together with MSX. Biochemical analysis revealed that R. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH+4 as well as by glutamate. The results demonstrate that utilization of solar energy to photoproduce large quantities of NH+4 from N2 is possible with photosynthetic bacteria by interfering with their regulatory control of N2 fixation.     

Nitrogen assimilation in facultative methylotrophic bacteria

A study was done of the pathways of nitrogen assimilation in the facultative methylotrophsPseudomonas MA andPseudomonas AM1, with ammonia or methylamine as nitrogen sources and with methylamine or succinate as carbon sources. When methylamine was the sole carbon and/or nitrogen source, both organisms possessed enzymes of the glutamine synthetase/glutamate synthase pathway, but when ammonia was the nitrogen sourcePseudomonas AM1 also synthesized glutamate dehydrogenase with a pH optimum of 9.0, andPseudomonas MA elaborated both glutamate dehydrogenase (pH optimum 7.5) and alanine dehydrogenase (pH optimum 9.0). Glutamate dehydrogenase and glutamate synthase from both organisms were solely NADPH-dependent; alanine dehydrogenase was NADH-dependent. No evidence was obtained for regulation of glutamine synthetase by adenylylation in either organism, nor did glutamine synthetase appear to regulate glutamate dehydrogenase synthesis.     

The regulation of glutamine transport and glutamine synthetase in Salmonella typhimurium.

Transport of glutamine by the high-affinity transport system is regulated by the nitrogen status of the medium. With high concentrations of ammonia, transport is repressed; whereas with Casamino acids, transport is elevated, showing behaviour similar to glutamine synthetase. A glutamine auxotroph, lacking glutamine synthetase activity, had elevated transport activity even in the presence of high concentrations of ammonia (and glutamine). This suggests that glutamine synthetase is involved in the regulation of the transport system. A mutant with low glutamate synthase activity had low glutamine transport and glutamine synthetase activities, which could not be derepressed. A mutant in the high-affinity glutamine transport system showed normal regulation of glutamate synthase and glutamine synthetase. Possible mechanisms for this regulation are discussed.     

Ammonia assimilation enzymes in a thermophilicBacillus sp. of marine origin

The physiology of ammonia assimilation enzymes was examined inBacillus sp. FE-1, a thermophilic marine bacterium. Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities varied with the nitrogen source present in the medium, ranging as much as 10-fold for the former and 2.5-fold for the latter. Glutamate dehydrogenase (GDH) was detected but, under the growth conditions studied, levels were not affected by the nitrogen source. Anaerobic growth in the presence of nitrate yielded enzyme levels that were not significantly different from those measured under aerobic growth. Partially purified GS exhibited a temperature optimum between 65° and 75°C. The enzyme's Mn²⁺-dependent reverse transferase activity was stimulated by K₂SO₄ and demonstrated some tolerance to NaCl. Hyperbolic kinetics were observed for ammonium, with an apparentK _M of 1.0mm.     

Enzymes of glutamine metabolism in testa-pericarp and endosperm of developing wheat grain

Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, glutamate puruvate transaminase and glutamate oxaloacetate transaminase have been assayed in developing testa-pericarp and endosperm of two wheat varieties, namely Shera (11.6% protein) and C-306 (9.8% protein). On per organ basis, activities of all the enzymes studied, except glutamine synthetase, increased during development. Glutamine synthetase activity decreased during development in the testa-pericarp, whereas, no glutamine synthetase activity could be detected in endosperm of either variety at any stage of development. Compared to testa-pericarp, endosperm had higher activities of glutamate synthase and glutamate pyruvate transaminase. On the whole, enzyme activities in Shera were higher, as compared to C-306. Developmental patterns and relative levels of enzyme activities in the two varieties were more or less the same, when expressed on dry weight basis or as specific activities. The results suggest that ammonia assimilation in developing wheat grain takes place by the glutamate dehydrogenase pathway in the endosperm; and both by the glutamate dehydrogenase and glutamine synthetase—glutamate synthase pathways in the testa-pericarp.     

What type of glutamine synthetase is important forStreptomyces coelicolor A3(2) under nitrogen-limited growth conditions?

Summary Glutamine synthetase I activity ofStreptomyces coelicolor was strongly repressed by ammonia and was induced 56.8 fold in a nitrogen-free medium. Glutamine synthetase II activity was not induced even by a long-term nitrogen starvation. Therefore, glutamine synthetase I is the only active enzyme ofStreptomyces coelicolor.