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|Glu, Gln, Asn, Gly, Ser|
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HORT640 - Metabolic Plant Physiology
Ammonia Assimilation and Recycling
Regulation of glutamine synthetase
Fungi and Yeasts
GS is encoded by the gene GLN1 in Saccharomyces cerevisiae (Mitchell, 1985).
GS is regulated by repression/derepression (mediated by glutamine). Mutants of the yeast Saccharomyces cerevisiae have been isolated which fail to derepress glutamine synthetase upon glutamine limitation. The mutations define a single nuclear gene, GLN3 (Mitchell and Magasanik, 1984b). The elevated NAD-GDH activity normally found in glutamate-grown cells is not found in gln3 mutants (Mitchell and Magasanik, 1984b). Glutamine limitation of gln1 structural mutants has the opposite effect, causing elevated levels of NAD-GDH even in the presence of ammonia (Mitchell and Magasanik, 1984b). A regulatory circuit that responds to glutamine availability through the GLN3 product has been proposed (Mitchell and Magasanik, 1984b).
Mitchell and Magasanik (1984c) propose that production of GS in Saccharomyces cerevisiae is controlled by three regulatory systems. One system responds to glutamine levels and depends on the positively acting GLN3 product (Mitchell and Magasanik, 1984c). The second system is general amino acid control, which couples derepression of a variety of biosynthetic enzymes to starvation for many single amino acids (Mitchell and Magasanik, 1984c). This system operates through the positive regulatory element GCN4. A third system responds to purine limitation (Mitchell and Magasanik, 1984c).
It is now known that in Saccharomyces cerevisiae, the transcription factors Gln3p and Nil1p of the GATA family play a determinant role in expression of genes that are subject to nitrogen catabolite repression (Soussi-Boudekou and Andre, 1999). In addition, the yeast mutant, gan1-1, exhibits dramatically decreased NAD-GDH and GS activities (Soussi-Boudekou and Andre, 1999). The GAN1 gene encodes a 488-amino-acid polypeptide bearing no typical DNA binding domain (Soussi-Boudekou and Andre, 1999). Gan1p is required for full expression of GLN1, GDH2 and also other nitrogen utilization genes, including GAP1, PUT4, MEP2 and GDH1 (Soussi-Boudekou and Andre, 1999).
In the food yeast Candia utilis GS is subject to cumulative feedback inhibition by end-products of glutamine metabolism in vitro, but this regulation was not demonstrable in vivo by direct measurements of the rate of glutamine synthesis (Sims and Ferguson, 1974).
In Candida utilis, GS is regulated by glutamine-mediated repression and reversible deactivation involving dissociation of active octomers into deactive tetramers (Ferguson and Sims, 1971; 1974b; Sims et al, 1974a). Sims and coworkers demonstrated a rapid inactivation of GS in Candida utilis on the addition of ammonia to glutamate-grown cultures. An increase in glutamine and a decrease in 2-oxoglutarate is implicated in this control (cf. glutamine/2-oxoglutarate ratio involvement in control of GS adenylation/deadenylation in gram-negative bacteria) (Ferguson and Sims, 1971; 1974b; Sims et al, 1974a). High glutamine concentrations promote the "relaxation" of the native 15.4 S enzyme into a 14.2 S octamer which dissociates reversibly into two 8.7 S tetramers. PEP promotes relaxation and formation of enzyme tetramers. NAD+, NADPH and ATP cause dissociation of tetramers into monomers. Glutamate and Mg2+ prevent dissociation and promote reassociation of tetramers (Sims et al, 1974b). Whereas 2-oxoglutarate can prevent dissociation of octamers it cannot promote reassociation (Sims et al, 1974b). Candida utilis GS tetramers have the same transferase activity as octamers, but have reduced synthetase activity. In the presence of 2-oxoglutarate and glutamate the enzyme can maintain its structural integrity under conditions which would otherwise lead to dissociation (Sims et al, 1974b).
In Saccharomyces cerevisiae GS is modulated by nitrogen repression and by two distinct inactivation processes (Legrain et al, 1982). Addition of glutamine to exponentially grown yeast leads to rapid enzyme inactivation that is reversed by removing glutamine from the growth medium (Legrain et al, 1982). A regulatory mutation (gdhCR mutation) suppresses this inactivation by glutamine in addition to its derepressing effect on enzymes involved in nitrogen catabolism (Legrain et al, 1982). The gdhCR mutation also increases the level of proteinase B in exponentially grown yeast (Legrain et al, 1982). Inactivation of GS is also observed during nitrogen starvation (Legrain et al, 1982). This inactivation is irreversible and consists very probably of a proteolytic degradation. Strains bearing proteinase A, B and C mutations are no longer inactivated under nitrogen starvation (Legrain et al, 1982).
The reversible in vivo inactivation of Saccharomyces cerevisiae GS by the addition of glutamine or ammonia is characterized by a specific loss of synthetase activity; transferase activity remains stable (Mitchell and Magasanik, 1984b). Several physiological perturbations cause inactivation, such as carbon starvation or limitation for a required amino acid, which could cause a buildup of glutamine (Mitchell and Magasanik, 1984b). In contrast to Candida utilis, no change in the native size of the enzyme was associated with inactivation of Saccharomyces cerevisiae GS, but there appears to be a change in the immunological properties of the enzyme subunit (Mitchell and Magasanik, 1984b).
Neurospora crassa GS is unusual in that it contains two non-identical polypeptides (Sanchez et al, 1980). When Neurospora crassa is grown exponentially on ammonium excess, ammonium is fixed by a glutamate dehydrogenase and an octameric glutamine synthetase (GS) (Lara et al, 1982). The synthesis of this GS polypeptide (beta) is regulated by the nitrogen source present in the medium; high on glutamate, intermediate on ammonium, and low on glutamine (Lara et al, 1982). However, when N. crassa is grown in fed-batch ammonium-limited cultures a different polypeptide of GS (alpha), arranged as a tetramer, is synthesized (Dunn-Coleman and Garrett, 1980; Lara et al, 1982). The tetrameric alpha GS is proposed to function with glutamate synthase in the assimilation of low ammonium concentrations (Lara et al, 1982). gln-1b mutant strains synthesize only the GS alpha monomer (i.e. lacks the GS beta monomer) (Calderon et al, 1990).
Calderon J, Martinez LM, Mora J 1990 Isolation and characterization of a Neurospora crassa mutant altered in the alpha polypeptide of glutamine synthetase. J. Bacteriol. 172: 4996-5000.
Dunn-Coleman NS, Garrett RH 1980 The role of glutamine synthetase and glutamine metabolism in nitrogen metabolite repression, a regulatory phenomenon in the lower eukaryote Neurospora crassa. Mol. Gen. Genet. 179: 25-32.
Ferguson AR, Sims AP 1971 Inactivation in vivo of glutamine synthetase and NAD-specific glutamate dehydrogenase: its role in the regulation of glutamine synthesis in yeasts. J. Gen. Microbiol. 69: 423-427.
Ferguson AR, Sims AP 1974a The regulation of glutamine metabolism in Candida utilis: the role of glutamine in the control of glutamine synthetase. J. Gen. Microbiol. 80: 159-171.
Ferguson AR, Sims AP 1974b The regulation of glutamine metabolism in Candida utilis: the inactivation of glutamine synthetase. J. Gen. Microbiol. 80: 173-185.
Lara M, Blanco L, Campomanes M, Calva E, Palacios R, Mora J 1982 Physiology of ammonium assimilation in Neurospora crassa. J. Bacteriol. 150: 105-112.
Legrain C, Vissers S, Dubois E, Legrain M, Wiame JM 1982 Regulation of glutamine synthetase from Saccharomyces cerevisiae by repression, inactivation and proteolysis. Eur. J. Biochem. 123: 611-616.
Mitchell AP 1985 The GLN1 locus of Saccharomyces cerevisiae encodes glutamine synthetase. Genetics 111: 243-258.
Mitchell AP, Magasanik B 1984a Regulation of glutamine-repressible gene products by the GLN3 function in Saccharomyces cerevisiae. Mol. Cell Biol. 4: 2758-2766.
Mitchell AP, Magasanik B 1984b Biochemical and physiological aspects of glutamine synthetase inactivation in Saccharomyces cerevisiae. J. Biol. Chem. 259: 12054-12062.
Mitchell AP, Magasanik B 1984c Three regulatory systems control production of glutamine synthetase in Saccharomyces cerevisiae. Mol. Cell Biol. 4: 2767-2773.
Sanchez F, Calva E, Campomanes M, Blanco L, Guzman J, Saborio JL, Palacios R 1980 Heterogeneity of glutamine synthetase polypeptides in Neurospora crassa. J. Biol. Chem. 255: 2231-2234.
Sims AP, Ferguson AR 1974 The regulation of glutamine metabolism in Candida utilis: studies with 15NH3 to measure in vivo rates of glutamine synthesis. J. Gen. Microbiol. 80: 143-158.
Sims AP, Toone J, Box V 1974a The regulation of glutamine synthesis in the food yeast Candida utilis: the purification and subunit structure of glutamine synthetase and aspects of enzyme deactivation. J. Gen. Microbiol. 80: 485-499.
Sims AP, Toone J, Box V 1974b The regulation of glutamine metabolism in Candida utilis: mechanisms of control of glutamine synthetase. J. Gen. Microbiol. 84: 149-162.
Soussi-Boudekou S, Andre B 1999 A co-activator of nitrogen-regulated transcription in Saccharomyces cerevisiae. Mol. Microbiol. 31: 753-762.
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