Regulation of glutamine synthetase in fed-batch cultures of Neurospora crassa

Limón-Lason, Jorge; Lara, Miguel; Resendiz, Bertha; Mora, Jaime

doi:10.1016/0006-291x(77)91425-5

Cited by 38 publications

(21 citation statements)

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“…Furthermore, 2-oxoglutarate is accumulated in this mutant strain (11,12). The relatively large incorporation of label into glutamate in the GDH-mutant strain (Table 1) can be explained as a result of high GOGAT activity and the accumulation of 2-oxoglutarate.…”

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13N isotope studies of glutamine assimilation pathways in Neurospora crassa

et al. 1989

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L-[amide-'3N]glutamine in Neurospora crassa is metabolized to [13N]glutamate by glutamate synthase and to [13N]ammonium by the glutamine transamina-w-amidase pathway. The [13Njammonium relased is assimilated by glutamate dehydrogenase and glutamine synthetase, confirming the operation of a glutamine cycle. Most of the nitrogen is retained during cycling between glutamate and glutamine.In Neurospora crassa, glutamine, in addition to participating in transamidation reactions, is converted to 2-oxoglutarate and ammonium by the enzymes of the glutamine transaminase-w-amidase pathway, in which a transaminase catalyzes transfer of amino nitrogen of glutamine to various oxo acids. 2-Oxoglutaramate thus produced is hydrolyzed by w-amidase to 2-oxoglutarate and ammonium ion (2).The amine nitrogen of glutamine is also assimilated by the action of glutamate synthase (glutamine oxoglutarate amidotransferase [GOGAT]), which synthesizes two molecules of glutamate from glutamine and 2-oxoglutarate (1). From the results obtained with several inhibitors and mutants, Calder6n and Mora concluded that the ammonium ion released by the w-amidase pathway is assimilated by glutamate dehydrogenase (GDH) and by glutamine synthetase (1, 2). These enzymes, when combined with the glutamine transaminase and w-amidase, yield a glutamine cycle in which glutamine is continually degraded and resynthesized (1). We report here the data obtained by using L-[amide-"3N]glutamine as a tracer to study the metabolic pathways of glutamine assimilation in N. crassa. 13N, the longest-lived radioisotope of nitrogen (t112 = 9.96 min), was used to label ammonia (7). L-[amide-'3N]glutamine was prepared from [13N]ammonia by using glutamine synthetase immobilized onto CNBr-activated Sepharose as previously described (16).The N. crassa cultures were grown for 8 h at 30°C on Vogel minimal medium (17) containing 1.5% sucrose and 25 mM NH4NO3 as the nitrogen source. Six milliliters of this medium was centrifuged, and after decantation, 50 ,ul of L-[amide-13N]glutamine (1 to 5 mCi [50 to 100 ,uM]) was added to the cell suspension. Uptake of label was stopped by adding 2 ml of water, and after centrifugation and a further wash, the metabolites were extracted with 0.2 ml of ice-cold 1% (wt/vol) picric acid. The '3N-labeled metabolites were analyzed by using a high-pressure liquid chromatography system (Series 4; The Perkin-Elmer Corp., Norwalk, Conn.) in which the effluent was continuously monitored with a Ramona D flowthrough y detector (5).A Partisil 10 SCX 10 ,uM (4.6 by 250 mm) (Phenomenex, Rancho Palo Verdes, Calif.) analytical cation-exchange column was used for separation; the elution conditions were as reported previously (5). In some cases glutamate was mea-* Corresponding author. sured by high-pressure liquid chromatography of the ophthaldialdehyde derivates (10), and ammonia content was determined enzymatically with glutamate dehydrogenase (4).The relative radioactivity incorporated into the a-amino and amide groups of glutamine was determined as follows. A...

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confidence: 89%

“…The difference in the labeling of the amine and the amide groups of glutamine ( The appreciable transfer of amine (glutamate plus glutamine) nitrogen to alanine in the GDH-strain (Table 1) but not in the wild-type strain may be explained by accumulation of pyruvate as a result of a decrease in 2-oxoglutarate utilization in this strain (11,14).…”

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confidence: 96%

13N isotope studies of glutamine assimilation pathways in Neurospora crassa

et al. 1989

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“…In studies on the primary nitrogen metabolism of fungi, yeasts and ascomycetes have been investigated preferentially (Brown et al, 1974;Casper et al, 1985;Dunn-Coleman et al, 198 1 ;Holmes et al, 1989;Kusnan et al, 1987Kusnan et al, ,1989Limon-Lason et al, 1977). In this paper the primary nitrogen metabolism of the basidomycetic fungus Stropharia semiglobata was investigated.…”

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confidence: 98%

The involvement of glutamate dehydrogenase and glutamine synthetase/glutamate synthase in ammonia assimilation by the basidiomycete fungus Stropharia semiglobata

Schwartz

Misri

Fock

1991

Journal of General Microbiology

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~When grown on ammonia as sole nitrogen source, the basidiomycete fungus Stropharia semigfobatu assimilated nitrogen via glutamate dehydrogenase (GDH). The two isoenzymes of GDH were both repressed by increasing concentrations of ammonia, but NADP-GDH (EC 1.4.1.4) was far more active than NAD-GDH (EC 1.4.1.2). Glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.7.1) were also active in S. semigfobatu and these enzyme activities were independent of the ammonia concentration in the suspension. From enzyme activity and lSN-labelling studies of amino acids, it is concluded that both GDH and GS/GOGAT contribute to ammonia assimilation in this fungus.

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“…Growth was monitored by measuring optical density at 650 nm. Fed-batch ammonium-limited cultures were achieved as previously described (14).…”

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Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity

Folch¹,

Antaramián²,

Rodríguez³

et al. 1989

J Bacteriol

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A mutant of Saccharomyces cerevisiae that lacks glutamate synthase (GOGAT) activity has been isolated. This mutant was obtained after chemical mutagenesis of a NADP-glutamate dehydrogenase-less mutant strain. The gdh gus mutant is a glutamate auxotroph. The genetic analysis of the gus mutant showed that the GOGAT-less phenotype is due to the presence of two loosely linked mutations. Evidence is presented which suggests the possibility that S. cerevisiae has two GOGAT activities, designated GOGAT A and GOGAT B. These activities can be distinguished by their pH optima and by their regulation by glutamate. Furthermore, one of the mutations responsible for the GOGAT-less phenotype affected GOGAT A activity, while the other mutation affected GOGAT B activity.Glutamate biosynthesis can be achieved through the reductive amination of 2-oxoglutarate catalyzed by NADP+-dependent glutamate dehydrogenase (NADP+-GDH) (2). In 1970, Tempest et al. (24) demonstrated the existence of an alternative pathway for glutamate biosynthesis in Klebsiella aerogenes. This pathway comprised glutamate synthase (GOGAT) and glutamine synthetase. The function of this pathway has been demonstrated in several microorganisms (3,12,23) and in higher plants (18). Physiological studies indicate that the main role of the glutamine synthetase-GOGAT pathway is in ammonium assimilation and glutamate biosynthesis under ammonium-limited conditions (23). In Neurospora crassa, GOGAT also appears to play an important role in glutamine degradation (4). N. crassa (21), Salmonella typhimurium (16), and Escherichia coli (5, 19) mutants lacking GOGAT activity have been obtained, and their characterization has contributed to the understanding of the role of GOGAT in glutamate biosynthesis.Although GOGAT activity has been previously detected in Saccharomyces cerevisiae (22), the role of GOGAT in glutamate biosynthesis and its regulation are poorly understood. Mutants altered in NADP+-GDH have been isolated from S. cerevisiae (7). The isolation of GOGAT-less mutants has been reported elsewhere (26); however, their genetic and physiological characterization has not been published, so the physiological function of GOGAT still remains obscure. Our results indicate that GOGAT plays a role in glutamate biosynthesis under conditions of ammonium excess and that under ammonium-limited conditions, both NADP+-GDH and GOGAT can participate in the synthesis of glutamate. We also present evidence suggesting that S. cerevisiae has two GOGAT activities.MATERIALS AND METHODS Strains. Table 1 describes the characteristics of the different strains used in this study.Growth conditions. Strains were routinely grown on minimal medium (MM) containing salts, trace elements, and vitamins following the formula of yeast nitrogen base (Difco * Corresponding author.Laboratories, Detroit, Mich.). Glucose (2% [wt/vol]) was used as the carbon source, and 40 mM (NH4)2SO4 was used as the nitrogen source. Amino acids needed to satisfy auxotrophic requirements were added at 0.01% (wt/vol). Cells wer...

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Regulation of glutamine synthetase in fed-batch cultures of Neurospora crassa

Cited by 38 publications

References 12 publications

13N isotope studies of glutamine assimilation pathways in Neurospora crassa

13N isotope studies of glutamine assimilation pathways in Neurospora crassa

The involvement of glutamate dehydrogenase and glutamine synthetase/glutamate synthase in ammonia assimilation by the basidiomycete fungus Stropharia semiglobata

Isolation and characterization of a Saccharomyces cerevisiae mutant with impaired glutamate synthase activity

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