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N Use By Plants
Nitrate Assimilation
Ammonia Assimilation
Glu, Gln, Asn, Gly, Ser
Aminotransferases
Asp, Ala, GABA
Val, Leu, Ileu, Thr, Lys
Pro, Arg, Orn
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HORT640 - Metabolic Plant Physiology

Ammonia Assimilation and Recycling

The glutamate synthase or GS-GOGAT cycle

For many years it was thought that bacteria and higher plants assimilate ammonia into glutamate via the GDH pathway, as in certain fungi and yeasts. However, in bacteria it became clear in 1970 that an alternative pathway of ammonia assimilation [involving glutamine synthetase (GS) [EC 6.3.1.2] and an NADPH-dependent glutamine:2-oxoglutarate amidotransferase (GOGAT) [EC 1.4.1.13] or glutamate synthase, must be operating when ammonia is present in the growth medium at low levels (Tempest et al, 1970). Thus, N-starvation leads to derepression and activation of GS (with a high affinity for NH3) and derepression of GOGAT, and repression of GDH (with a relatively low affinity for NH3) (Tempest et al, 1970). High ammonia availability leads to repression and deactivation of GS and induction of GDH (Tempest et al, 1970).

GDH
NH3 + 2-oxoglutarate + NADPH + H+ <---> glutamate + NADP+

GS-GOGAT
NH3 + glutamate + ATP ---> glutamine + ADP + Pi
glutamine + 2-oxoglutarate + NADPH + H+ ---> 2 glutamate + NADP+

Both the GDH and GS-GOGAT pathways produce 1 mole of glutamate from 1 mole each of NH3, 2-oxoglutarate and NADPH. But note that the GS-GOGAT pathway is energetically more costly than the GDH pathway, consuming 1 ATP.

Escherichia coli is now known to have two primary pathways for glutamate synthesis (Hellig, 1994; 1998). The GS-GOGAT pathway is essential for glutamate synthesis at low ammonium concentrations and for regulation of the glutamine pool, and is used when the cell is not under energy limitation (Hellig, 1994; 1998). The GDH pathway is used in glutamate synthesis when the cell is limited for energy (and carbon; i.e. glucose-limited growth) but ammonium and phosphate are present in excess (Hellig, 1994; 1998). Synechocystis sp. strain PCC 6803 utilizes the GS-GOGAT pathway as the primary pathway of ammonia assimilation, but the presence of GDH appears to offer a selective advantage for the cyanobacterium under nonexponential growth conditions (Chavez et al, 1999). These dual pathways may be common to bacteria, cyanobacteria, algae, yeasts and fungi (Huth and Liebs, 1988).

Re-examination of ammonia assimilation in yeasts and fungi now reveals the operation of alternative pathways of glutamate synthesis, independent of NADPH-GDH:

  • Mutants of Aspergillus nidulans lacking NADP-GDH activity grow more poorly than wild-type strains on ammonium as a sole nitrogen source (Macheda et al, 1999). The leaky growth of these mutants is indicative of an alternative pathway of ammonium assimilation and glutamate biosynthesis (Macheda et al, 1999). A. nidulans mutants disrupted in the gltA encoding GOGAT, were found to be dispensable for growth on ammonium in the presence of NADP-GDH (Macheda et al, 1999). However, a strain carrying the gltA inactivation together with an NADP-GDH structural gene mutation (gdhA) was unable to grow on ammonium or on nitrogen sources metabolized via ammonium (Macheda et al, 1999).

  • Schizosaccharomyces pombe mutants lacking either NADPH-GDH or GOGAT are still able to grow on ammonium as sole nitrogen source (Perysinakis et al, 1995). Complete lack of growth on ammonium as sole N source is seen only in double mutants lacking both NADPH-GDH and GOGAT (Perysinakis et al, 1995).

  • In contrast to Candida utilis (Sims and Folkes, 1964), analysis of 15N-ammonium assimilation in actively growing mycelium of Agaricus bisporus indicates participation of the GS-GOGAT pathway, and no participation of NADP-GDH (Baars et al, 1996). 13NH3 tracer studies indicate that the GS-GOGAT pathway is the major route of ammonium assimilation in Candida albicans and also in nitrogen-starved cultures of Saccharomyces cerevisiae and Candida tropicalis (Holmes et al, 1989; 1991).

The yeast Saccharomyces cerevisiae synthesizes glutamate through the action of either NADP-glutamate dehydrogenase (NADP-GDH), encoded by GDH1 (under conditions of ammonia excess), or through the combined action of glutamine synthetase (GS) and glutamate synthase (GOGAT), encoded by GLN1 and GLT1 (under conditions of ammonia limitation) (Avendano et al, 1997). Dynamic modeling indicates that the GS-GOGAT pathway plays a more important physiological role in yeast than is generally assumed (van Riel et al, 1998). However, a double mutant of S. cerevisiae lacking NADP-GDH and GOGAT activities was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through a third pathway (Avendano et al, 1997). A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome led to the discovery that GDH1 showed high identity to an open reading frame (GDH3) on chromosome I (Avendano et al, 1997). Triple mutants impaired in GDH1, GLT1, and GDH3 are strict glutamate auxotrophs, indicating that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired (Avendano et al, 1997). This appears to be the first example of a microorganism possessing three pathways for glutamate biosynthesis (Avendano et al, 1997).

Following the discovery of glutamate synthase (GOGAT) in bacteria, a similar activity was sought in plants. A ferredoxin-dependent glutamate synthase [EC 1.4.7.1] was discovered in photosynthetic tissues of higher plants in 1974 (Lea and Miflin, 1974), and an NADH-dependent "glutamate synthetase" in non-photosynthetic plant tissues in the same year (Fowler et al, 1974).

Evidence in favor of the operation of the GS-GOGAT cycle as the primary pathway of ammonia assimilation in higher plants has been reviewed by several authors (e.g. Miflin and Lea, 1980; Lea et al, 1992). This evidence includes:

  • almost complete inhibition of 15NH4+ assimilation by the glutamine synthetase (GS) inhibitor, methionine sulfoximine (MSX) (Miflin and Lea, 1980; Lea et al, 1992).
  • quantitative analysis of 15NH4+ in Lemna minor is consistent with incorporation of 15N primarily into glutamine-amide, followed by transfer to glutamate and the amino-group of glutamine via the action of GOGAT and GS, respectively, provided that it is assumed that the GS-GOGAT cycle is compartmentilized in the chloroplast, and that a second site of glutamine synthesis occurs in the cytoplasm (Rhodes et al, 1980).
  • the maize gdh1-null mutant exhibits about 5% of the total GDH enzyme activity of wildtype plants. Although this mutant exhibits a slightly reduced total rate of 15NH4+ assimilation, when methionine sulfoximine (MSX), a potent inhibitor of GS is supplied, this completely blocks 15NH4+ assimilation in both the mutant and wildtype roots and shoots (Magalhaes et al, 1990). The contribution of GDH to net ammonia assimilation is small in comparison to that catalyzed by the GS-GOGAT cycle (Rhodes et al, 1989; Magalhaes et al, 1990).
  • mutants of Arabidopsis and barley defective in GS or GOGAT exhibit markedly impaired ammonia assimilation, especially under photorespiratory conditions (Lea et al, 1992).

Enzyme kinetic considerations also suggest a role for the GS-GOGAT pathway in ammonia assimilation at low tissue/cell ammonia concentrations. GS has a much higher affinity for ammonia than GDH and is viewed as a scavenger of ammonia in bacteria (Tempest et al, 1970) and in plants (see e.g. Miflin and Lea, 1980; Lea et al, 1992).

The major role of GDH in tissue cultured cells is the oxidation of glutamate to provide sufficient carbon skeletons for effective functioning of the TCA cycle (Robinson et al, 1991).

In wildtype Arapidopsis, GDH1 mRNA accumulates to high levels in dark-adapted or sucrose-starved plants; light or sucrose treatment each repress GDH1 mRNA accumulation. These results suggest that the GDH1 gene product functions in the direction of glutamate catabolism under carbon-limiting conditions (Melo-Oliveira et al, 1996). Low levels of GDH1 mRNA present in leaves of light-grown plants can be induced by exogenously supplied ammonia (Melo-Oliveira et al, 1996). Under such conditions of carbon and ammonia excess, GDH1 may function in the direction of glutamate biosynthesis (Melo-Oliveira et al, 1996). The recessive Arabidopsis glutamate dehydrogenase-deficient mutant allele gdh1-1 cosegregates with the GDH1. The gdh1-1 mutant displays a conditional phenotype; seedling growth is specifically retarded on media containing exogenously supplied inorganic nitrogen, suggesting that GDH1 plays a nonredundant role in ammonia assimilation under conditions of inorganic nitrogen excess (Melo-Oliveira et al, 1996). This is consistent with the fact that the levels of mRNA for GDH1 and chloroplastic glutamine synthetase (GS2) are reciprocally regulated by light (Melo-Oliveira et al, 1996).

see also: GS-GOGAT Cycle Simulation

References

Avendano A, Deluna A, Olivera H, Valenzuela L, Gonzalez A 1997 GDH3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae. J. Bacteriol. 179: 5594-5597.

Baars JJ, Op den Camp HJ, van der Drift C, Joordens JJ, Wijmenga SS, van Griensven LJ, Vogels GD 1996 15N-NMR study of ammonium assimilation in Agaricus bisporus. Biochim. Biophys. Acta 1310: 74-80.

Chavez S, Lucena JM, Reyes JC, Florencio FJ, Candau P 1999 The presence of glutamate dehydrogenase is a selective advantage for the cyanobacterium Synechocystis sp. strain PCC 6803 under nonexponential growth conditions. J. Bacteriol. 181: 808-813.

Fowler MW, Jessup W, Sarkissian GS 1974 Glutamate synthetase type activity in higher plants. FEBS Letts. 46: 340-342.

Helling RB 1994 Why does Escherichia coli have two primary pathways for synthesis of glutamate? J. Bacteriol. 176: 4664-4668.

Helling RB 1998 Pathway choice in glutamate synthesis in Escherichia coli. J. Bacteriol. 180: 4571-4575.

Holmes AR, Collings A, Farnden KJ, Shepherd MG 1989 Ammonium assimilation by Candida albicans and other yeasts: evidence for activity of glutamate synthase. J. Gen. Microbiol. 135: 1423-1430.

Holmes AR, McNaughton GS, More RD, Shepherd MG 1991 Ammonium assimilation by Candida albicans and other yeasts: a 13N isotope study. Can. J. Microbiol. 37: 226-232.

Huth J, Liebs P 1988 Nitrogen regulation in microorganisms. Zentralbl. Mikrobiol. 143: 179-194.

Lea PJ, Blackwell RD, Joy KW 1992 Ammonia assimilation in higher plants. In (K Mengel, DJ Pilbeam eds) "Nitrogen Metabolism of Plants", Clarendon Press, Oxford, pp 153-186.

Lea PJ, Miflin BJ 1974 An alternative route for nitrogen assimilation in higher plants. Nature (Lond.) 251: 614-616.

Macheda ML, Hynes MJ, Davis MA 1999 The Aspergillus nidulans gltA gene encoding glutamate synthase is required for ammonium assimilation in the absence of NADP-glutamate dehydrogenase. Curr. Genet. 34: 467-471.

Magalhaes JR, Ju GC, Rich PJ, Rhodes D 1990 Kinetics of 15NH4+ assimilation in Zea mays: Preliminary studies with a glutamate dehydrogenase (GDH1) null mutant. Plant Physiol. 94: 647-656.

Melo-Oliveira R, Oliveira IC, Coruzzi GM 1996 Arabidopsis mutant analysis and gene regulation define a nonredundant role for glutamate dehydrogenase in nitrogen assimilation. Proc. Natl. Acad. Sci. U.S.A. 93: 4718-4723.

Miflin BJ, Lea PJ 1980 Ammonia assimilation. In (BJ Miflin ed) "The Biochemistry of Plants" Vol 5, Academic Press, New York, pp 169 - 202.

Perysinakis A, Kinghorn JR, Drainas C 1995 Glutamine synthetase/glutamate synthase ammonium-assimilating pathway in Schizosaccharomyces pombe. Curr. Microbiol. 30: 367-372.

Rhodes D, Sims AP, Folkes BF 1980 Pathway of ammonia assimilation in illuminated Lemna minor. Phytochem. 19: 357-365.

Robinson SA, Slade AP, Fox GG, Phillips R, Ratcliffe RG, Stewart GR 1991 The role of glutamate dehydrogenase in plant nitrogen metabolism. Plant Physiol. 95: 509-516.

Sims AP, Folkes BF 1964 A kinetic study of the assimilation of 15N-ammonia and the synthesis of amino acids in an exponentially growing culture of Candida utilis. Proc. Roy. Soc. Lond. B. Biol. Sci. 159: 479-502.

Tempest DW, Meers JL, Brown CM 1970 Synthesis of glutamate in Aerobacter aerogenes by a hitherto unknown route. Biochem. J. 117: 405-407.

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