Pathway Of Ammonia Assimilation Research Articles

Evidence for ammonia as an inhibitor of heterocyst and nitrogenase formation in the cyanobacterium Anabaena cycadeae

Growth and regulation of heterocyst and nitrogenase by fixed nitrogen sources were studied comparatively in parent and glutamine auxotrophic mutant of Anabaena cycadeae. The parent strain grew well on N2, NH+4 or glutamine while the mutant strain grew on glutamine but not on N2 or NH+4. The total lack of active glutamine synthetase in the mutant strain thus appears to be the reason for its observed lack of growth in N2 or NH+4, which explains why it is a glutamine auxotroph and at the same time shows glutamine synthetase to be the sole primary ammonia assimilating enzyme. NH+4 repression of heterocyst and nitrogenase in the mutant and the parental strains and their derepression by L-methionine-DL-sulfoximine suggest that NH+4 per se and not glutamine synthetase mediated pathway of ammonia assimilation is the initial repressor signal of heterocyst and nitrogenase in A. cycadeae.

13N isotope studies on the pathway of ammonia assimilation in Bacillus megaterium and Escherichia coli

The pathway of ammonia incorporation into amino acids was studied by use of 13N-ammonium ions in Bacillus megaterium and Escherichia coli that had been grown aerobically on a minimal salts medium containing NH4Cl as the source of nitrogen. Anion- and cation-exchange high-pressure-liquid chromatography was used to separate amino acids relevant to the several possible pathways for ammonia assimilation in bacteria. At an initial concentration of added NH4+ of 1 microM, the glutamine synthetase-glutamate synthase pathway represented the major pathway in both bacteria on the basis of the effects of inhibitors of that pathway (L-methionine-DL-sulfoximine and azaserine) and of transamination (aminooxy-acetate) and the observation that the specific activity of glutamine was greater initially than that of any other amino acid likely to be the first product of an assimilation pathway. The study provides (i) a new analytical method for 13N-tracer investigation of amino acids, (ii) confirmation of conclusions from enzymological studies on the pathway of ammonia assimilation in B. megaterium and E. coli, and (iii) proof that alanine dehydrogenase and aspartate ammonia lyase (aspartase) are not important pathways in B. megaterium at low NH4+ concentrations.

Sources of Ammonium in Oat Leaves Treated with Tabtoxin or Methionine Sulfoximine

Excised 7-day-old oat (Avena sativa L. cv. Jaycee) leaves were incubated in media containing 7.1 millimolar KNO(3) and 0.15 millimolar tabtoxin or 1 millimolar methionine sulfoximine (MSO) to investigate the sources of the observed ammonium accumulated. Tabtoxin and MSO are known inhibitors of glutamine synthetase, the first enzyme in the primary pathway of ammonium assimilation. During a 4- to 6-hour incubation, there was little net change in protein or total amino acid concentration. Alanine, aspartate/asparagine, and glutamate/glutamine decreased markedly under these treatments, whereas several other amino acids increased. Exogenous (15)N from K(15)NO(3) was taken up and incorporated into the nitrate and ammonium fractions of leaves treated with tabtoxin or MSO. This result and the high in vitro activities of nitrate reductase indicated that reduction of nitrate was one source of the accumulated ammonium. Leaves incubated under 2% O(2) to reduce photorespiration accumulated only about 13% as much ammonium as did those under normal atmospheres. We conclude that most of the tabtoxin- or MSO-induced ammonium came from photo-respiration, and the remainder was from nitrate reduction.

Enzymes of ammonia assimilation in root nodules of Trigonella foenum-graecum

The main pathway of ammonia assimilation in the root nodules of Trigonella foenum-graecum is via nodule cytosol glutamine synthetase-glutamate synthase.

Primary assimilation process of triply (15N,14C and3H) labeled arginine in the roots of arginine-fed barley

Abstract To clarify the mechanism of arginine utilization in barley roots, triply labeled (ureido-15N, ureido-14C and 5-3H)arginine was applied to plants precultured with arginine (Arg-plants). (5 -3H)Arginine was incorporated mainly into ornithine, suggesting that arginase contributes in the first step of arginine metabolism. The arginase activity in the tissues was greatly enhanced by continuous supply of arginine, whereas urease activity was not by the same treatment. The amount of 14CO2 evolved from (ureido-14C)arginine in the Arg-plants was several times higher than that in plants treated with NO3 - (NO3-plants), and most 14C-urea exogenously supplied to detached roots of Arg-plants was immediately decomposed to 14CO2. The urea released from arginine by arginase was cleaved to 15NH4 ++14CO2 by urease. Most of the 14CO2 was then lost from the root system. On the other hand, the released 15NH4 + was reassimilated into amino acids probably through the pathway of ammonia assimilation. Released (5-3H)ornithine was metabolized dominantly to proline.

Pathway of ammonia assimilation in illuminated and darkened Chlamydomonas reinhardii

Evidence is presented which shows that NH3 assimilation in Chlamydomonas occurs exclusively via the glutamate synthase cycle in illuminated and darkened cells and those in which the internal level of NH3 is elevated. This result indicates that glutamate dehydrogenase probably plays a catabolic rather than anabolic role in the N nutrition of the alga. Glutamine synthetase and glutamate dehydrogenase were characterized and their kinetic properties shown to be consistent with these proposals. It is suggested that reversible activity modulations of glutamine synthetase regulate the operation of the glutamate synthase cycle in the light but the availability of reductant and ATP limits its activity in darkened cells. The possible involvement of the two glutamate synthase enzymes in both light and dark assimilation is discussed.

Pathway of ammonia assimilation in illuminated Lemna minor

Plants of duckweed ( Lemna minor) were grown under constant illumination and with a controlled supply of ammonium-N so as to maintain a constant low concentration. In two kinetic experiments (differing in illumination and N level) with 15N-ammonia, plants were periodically harvested and their free amino acids analysed for 15N abundance. Attempts were then made to fit the data by computer simulation models. Only models which had at least two or more intracellular compartments gave adequate fits. Two two-compartment models were tested fully. Both had in compartment 1 the glutamine synthetase-glutamate synthase cycle and in compartment 2 a second site of glutamine synthesis. In one model the glutamate for compartment 2 was derived by transport from compartment 1; in the second model it was synthesized from ammonia by glutamate dehydrogenase at a rate equivalent to 10% of the total N uptake. This second model was rejected after it was found that plants previously treated with methionine sulphoximine and aza-serine (inhibitors of the glutamate synthase cycle) were unable to incorporate 15N. In spite of wide differences in labelling pattern between the two experiments the first model gave acceptable fits to both when different pool sizes were allowed for. Operation of the glutamate synthase cycle was confirmed by the correspondence between model and data for labelling of glutamine amide, glutamine amino and glutamic acid. Consideration of enzyme distributions suggested that compartment 1 (the glutamate synthase system) is the chloroplasts and compartment 2 the cytosol. Analysis of asparagine and neutral amino acids made it possible to construct balance sheets for N uptake in the two experiments. They suggest that all glutamine synthesized in the chloroplast is used for glutamate and asparagine synthesis and that the cytosol enzyme meets the need of the cell for glutamine per se. The high turnover rates for asparagine indicate that this compound is an important intermediate even under steady state conditions, and carries between 20 and 50% of the products of N assimilation.

Pathways of ammonia assimilation in obligate methane utilizers

Pathways of ammonia assimilation in obligate methane utilizers

Pathways of ammonia assimilation in obligate methane utilizers

Pathways of ammonia assimilation in obligate methane utilizers

Glutamine Synthetase/Glutamine: α-Ketoglutarate Aminotransferase in Chloroplasts from the Marine Alga Caulerpa simpliciuscula

The enzymic capacities for ammonia assimilation into amino acids have been investigated in chloroplasts from the siphonous green alga Caulerpa simpliciuscula (Turner) C. Ag. The results show that these chloroplasts differ from those of higher plants in having present simultaneously the enzymic capacities to permit assimilation of ammonia by two pathways. Glutamine synthetase (EC 6.3.1.2) activity at levels up to 4 mumoles per mg chlorophyll per hour were found in soluble extracts of the chloroplasts. Glutamine(amide):alpha-ketoglutarate aminotransferase (oxidoreductase ferredoxin) (EC 1.4.7.1) activity at levels up to 1.4 mumoles per mg chlorophyll per hour was detected by incubation of photosynthetically active chloroplasts either in light or with reduced ferredoxin. Together these enzymes provide the capacity for the conventional pathway of ammonium assimilation in chloroplasts via glutamine. A similar level of a glutamate dehydrogenase with an unusually low K(m) for ammonia which has been described previously in these chloroplasts provides the second potential pathway.

Enzymes of Nitrogen Metabolism in Legume Nodules: a Comparative Study

Levels of activity of glutamine synthetase, glutamate dehydrogenase and NADH-dependent glutamate synthase in nodule cytoplasm extracts of twelve herbaceous legume species have been measured. Nodules of all species contained substantial quantities of glutamine synthetase. Levels of glutamate synthase were found to be between 7 and 100% of those of glutamine synthetase, while levels of glutamate dehydrogenase varied widely between 0.2 and 150% of those of glutamine synthetase. The estimated Km for hydroxylamine of glutamine synthetase was found to vary between 0.02 and 0.5 mM in nine species tested, while that of glutamate dehydrogenase for ammonia varied between 0.03 M and 0.1 M in the four species containing significant levels of that enzyme. It is proposed that the pathway of ammonia assimilation via glutamine synthetase and NADH-dependent glutamate synthase-catalysed reactions is universal in legume nodule metabolism.

Mechanism of ammonia assimilation in streptococci.

SUMMARY: Streptococcus sanguis, S. bovis, S. mutans and S. salivarius, and also Escherichia coli for comparison, were grown separately in a chemostat under ammonia- and glucose-limitation. Bacterial extracts were assayed for ammonia-assimilating enzymes. For E. coli the level of glutamate dehydrogenase decreased and the levels of glutamine synthetase and glutamine (amide): 2-oxoglutarate aminotransferase (glutamate synthase) increased following a change from glucose- to ammonia-limitation. In contrast, ammonia-limited streptococci contained much higher levels of glutamate dehydrogenase than glucose-limited streptococci, and although a glutamine synthetase was detected in S. bovis, glutamate synthase could not be detected in any of the streptococci. Low activity of a NAD-linked alanine dehydrogenase was found in S. bovis and S. sanguis. These results suggest that the only pathway of ammonia assimilation in streptococci involves glutamate dehydrogenase, irrespective of the nature of the growth limitation. Glutamate dehydrogenase extracted from the different species of Streptococcus had similar Michaelis constants and optimum pH values.

The mechanism of ammonia assimilation in nitrogen fixing Bacteria.

Enzymatic and genetic evidence are presented for a new pathway of ammonia assimilation in nitrogen fixing bacteria: ammonium → glutamine → glutamate. This route to the important glutamate-glutamine family of amino acids differs from the conventional pathway, ammonium → glutamate → glutamine, in several respects. Glutamate synthetase [(glutamine amide-2-oxoglutarate aminotransferase) (oxidoreductase)], which is clearly distinct from glutamate dehydrogenase, catalyzes the reduced pyridine nucleotide dependent amination of α-ketoglutarate with glutamine as amino donor yielding two molecules of glutamate as product. The enzyme is completely inhibited by the glutamine analogue DON, whereas glutamate dehydrogenase is not affected by this inhibitor; the glutamate synthetase reaction is irreversible. Glutamate synthetase is widely distributed in bacteria; the pyridine nucleotide coenzyme specificity of the enzyme varies in many of these species. The activities of key enzymes are modulated by environmental nitrogenous sources; for example, extracts of N2-grown cells of Klebsiella pneumoniae form glutamate almost exclusively by this new route and contain only trace amounts of glutamate dehydrogenase activity whereas NH3-grown cells possess both pathways. Also, the biosynthetically active form of glutamine synthetase with a low K m for ammonium predominates in the N2-grown cell. Several mutant strains of K. pneumoniae have been isolated which fail to fix nitrogen or to grow in an ammonium limited environment. Extracts of these strains prepared from cells grown on higher levels of ammonium have low levels of glutamate synthetase activity and contain the biosynthetically inactive species of glutamine synthetase along with high levels of glutamate dehydrogenase. These mutants missing the new assimilatory pathway have serious defects in their metabolism of many inorganic and organic nitrogen sources; utilization of at least 20 different compounds is effected. We conclude that the new ammonia assimilatory route plays an important role in nitrogenous metabolism and is essential for nitrogen fixation.

A KINETIC STUDY OF THE ASSIMILATION OF (15N)-AMMONIA AND THE SYNTHESIS OF AMINO ACIDS IN AN EXPONENTIALLY GROWING CULTURE OF CANDIDA UTILIS.

Ammonium phosphate labelled with $^{15}$N has been used in a single quantitative experiment to trace the pathways of ammonia assimilation and amino acid synthesis in food yeast. Methods have been developed and are briefly described, whereby the free amino acids and amides, and the amino acid residues of the proteins, may be extracted from the yeast, separated by ion-exchange chromatography, quantitatively estimated, and the nitrogen of their $\alpha $-amino groups specifically liberated for isotopic analysis.The yeast was cultured in shake-flasks on a minimal medium containing glucose, ammonium phosphate and mineral salts. By analysis of cells removed from the culture at various times it was shown that the percentage composition remained sensibly constant throughout the part of the exponential phase investigated, and hence the yeast was assumed to be in steady-state growth. For the isotopic experiment the yeast culture was transferred to medium containing ($^{15}$NH$_{4}$)$_{2}$HPO$_{4}$ and samples where then removed at intervals for analysis of the free and protein amino acids and for measurement of their $^{15}$N-abundance. After 30 min the remaining yeast was transferred back into unlabelled medium and further samples were then taken. This double-transfer procedure was used in order to permit more stringent tests of metabolic relationships to be made in the subsequent kinetic analysis.The quantitative analysis of the isotopic data was made by comparison with a model reaction system. The model consists of a series of branching reaction chains linking steady-state pools of intermediates from which material is randomly withdrawn in subsequent reactions; primary products of nitrogen assimilation can give rise to secondary and tertiary derivatives which, as amino acids, can act as precursors in protein synthesis. A series of kinetic equations have been derived, relating the isotopic abundance of a component in the model to the rates of the various reactions involved in its biosynthesis. By substituting numerical values in these equations and comparing the results with the experimental data it has been possible to assign to each amino acid a position in the model and to make an estimate of its rate of synthesis. This estimate can then, as a further test, be compared with the rate known to be necessary to maintain steady-state growth.The kinetic analysis indicates that glutamic acid and glutamine are the only amino acids to derive their $\alpha $-nitrogen directly from ammonia; they are synthesized at a rate sufficient to provide all the $\alpha $-amino nitrogen required for growth of the yeast but not to meet the total nitrogen requirements, so that other pathways for the assimilation of nitrogen must also operate. All the other amino acids apparently derive their $\alpha $-amino-N from glutamic acid, many of them directly. For some of the amino acids, the labelling of the residues in the protein is consistent with their having come directly from the pool of free amino acid; this emphasizes the very small size of any pools of intermediates between amino acid and protein. For other amino acids a more complex relationship has been observed between the free amino acid pool and the proteins; the data are best interpreted by assuming that not all of the pool is available as an intermediate in protein synthesis, some of it being spatially separated and not further metabolized. This separate pool is here called a storage pool and is envisaged as functioning as part of the regulatory mechanisms of the cell by removing any small overproduction of amino acid.The results are further considered in relation to known pathways of amino acid biosynthesis in micro-organisms. The data for alanine, aspartic acid, glycine, leucine, isoleucine, valine, tyrosine and phenylalanine are consistent with these amino acids, having been formed directly by transamination from glutamic acid. Similar transaminations, but followed by other reactions, can account for the synthesis of histidine, lysine, serine and methionine; there is no evidence for alanine-hydroxypyruvate transamination in serine synthesis or for the operation of the cystathionine pathway to methionine. Threonine does not apparently derive its nitrogen from aspartic acid in this experiment, and the operation of the pathway from aspartic acid via homoserine to threonine is questioned for yeast grown on a minimal medium. The very low isotopic abundance in free ornithine suggests that this amino acid pool, or at least 97% of it, is not an intermediate in arginine synthesis. Other mechanisms for the formation of citrulline and arginine are put forward. Proline is apparently formed from glutamic acid. The results are generally at variance with the concept of amino acid families proposed by the Carnegie Institution group; with the possible exception of the glutamic acid family there is no evidence for the transfer of nitrogen from the family head to member amino acids. It is suggested therefore that these are really keto acid families and that transamination reactions are of major importance in amino acid biosynthesis from inorganic nitrogen.