Glutamine assimilation pathways in Neurospora crassa growing on glutamine as sole nitrogen and carbon source

Neurospora crassa wild-type is almost unable to grow on glutamine as sole nitrogen and carbon source but a GDH-; GS +/- double mutant strain, lacking NADP-dependent glutamate dehydrogenase and partially lacking glutamine synthetase did grow. Under these conditions, the double mutant had a higher chemical energy content than the wild-type. Enzyme assays and labelling experiments with glutamine indicated that in the double mutant glutamine was degraded to ammonium and to carbon skeletons by glutamate synthase, the catabolic (NADH-dependent) glutamate dehydrogenase and the glutamine transaminase-omega-amidase pathway.     

Oxidation of Neurospora crassa glutamine synthetase.

The glutamine synthetase of Neurospora crassa, either purified or in cell extracts, was inactivated by ascorbate plus FeCl3 and by H2O2 plus FeSO4. The inactivation reaction was oxygen dependent, inhibited by MnCl2 and EDTA, and stimulated in cell extracts by sodium azide. This inactivation could also be brought about by adding NADPH to the cell extract. The alpha and beta polypeptides of the active glutamine synthetase were modified by these inactivating reactions, giving rise to two novel acidic polypeptides. These modifications were observed with the purified enzyme, with cell extracts, and under in vivo conditions in which glutamine synthetase is degraded. The modified glutamine synthetase was more susceptible to endogenous phenylmethylsulfonyl fluoride-insensitive proteolytic activity, which was inhibited by MnCl2 and stimulated by EDTA. The possible physiological relevance of enzyme oxidation is discussed.     

Physiology of ammonium assimilation in Neurospora crassa.

In Neurospora crassa the assimilation of high and low concentrations of ammonium occurs by two different pathways. When the fungi are growing exponentially on ammonium excess, this compound is fixed by a glutamic dehydrogenase and an octameric glutamine synthetase (GS). The synthesis of this GS polypeptide (beta) is regulated by the nitrogen source present in excess; being higher on glutamate, intermediate on ammonium, and lower on glutamine. When N. crassa is growing in fed-batch ammonium-limited cultures a different polypeptide of GS (alpha), arranged as a tetramer, is synthesized. In both conditions synthesis in vivo correlates with the data obtained with an in vitro translation system primed with N. crassa RNA. This different expression of alpha and beta GS polypeptides was also observed when the cultures were shifted from excess to low nitrogen, and vice versa. By agarose gel electrophoresis in the presence of methylmercury hydroxide, some separation of different mRNAs that direct the in vitro synthesis of alpha and beta GS polypeptides has been accomplished. Data are presented that establish the operation of the tetrameric alpha GS and of glutamate synthase in the assimilation of ammonium in low concentration.     

Neurospora crassa mutant impaired in glutamine regulation.

The final products of the catabolism of arginine that can be utilized as nitrogen sources by Neurospora crassa are ammonium, glutamic acid, and glutamine. Of these compounds, only glutamine represses arginase and glutamine synthetase. We report here the isolation and characterization of a mutant of N. crassa whose arginase, glutamine synthetase, and amino acid accumulations are resistant to glutamine repression (glnI). This mutant has a greater capacity than the wild type (glns) to accumulate most of the arginine and some of the glutamine in osmotically sensitive compartments while growing exponentially. Nonetheless, the major part of the glutamine remains soluble and metabolically available for repression. We propose that the lower repression of glutamine synthetase by glutamine in this mutant could be a necessary condition for sustaining the higher flow of nitrogen for the accumulation of amino acids observed in ammonium excess and that, if glutamine is the nitrogen signal that regulates the arginine accumulation of the vesicle, the glnr mutant has also escaped this control. Finally, in the glnr mutant, some glutamine resynthesis is necessary for arginine biosynthesis and accumulation.     

Nitrogen regulation of glutamine synthetase in Neurospora crassa.

A higher activity of glutamine synthetase (EC 6.3.1.2) was found in Neurospora crassa when NH4+ was limiting as nitrogen source than when glutamate was limiting. When glutamate, glutamine or NH4+ were in excess, a lower activity was found. Immunological titration and sucrose gradient sedimentation of the enzyme established that under all these conditions enzyme activity corresponded to enzyme concentration and that the octamer was the predominant oligomeric form. When N. crassa was shifted from nitrogen-limiting substrates to excess product as nitrogen source, the concentration of glutamine synthetase was adjusted with kinetics that closely followed dilution by growth. When grown on limiting amounts of glutamate, a lower oligomer was present in addition to the octameric form of the enzyme. When the culture was shifted to excess NH4+, glutamine accululated at a high rate; nevertheless, there was only a slow decrease in enzyme activity and no modification of the oligomeric pattern.     

Immunochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs.

Glutamine synthetase derived from two Neurospora crassa glutamine auxotrophs was characterized. Previous genetic studies indicated that the mutations responsible for the glutamine auxotrophy are allelic and map in chromosome V. When measured in crude extracts, both mutant strains had lower glutamine synthetase specific activity than that found in the wild-type strain. The enzyme from both auxotrophs and the wild-type strain was partially purified from cultures grown on glutamine as the sole nitrogen source, and immunochemical studies were performed in crude extracts and purified fractions. Quantitative rocket immunoelectrophoresis indicated that the activity per enzyme molecule is lower in the mutants than in the wild-type strain; immunoelectrophoresis and immunochemical titration of enzyme activity demonstrated structural differences between the enzymes from both auxotrophs. On the other hand, the monomer of glutamine synthetase of both mutants was found to be of a molecular weight similar to that of the wild-type strain. These data indicate that the mutations are located in the structural gene of N. crassa glutamine synthetase.     

Genetics and physiology of Neurospora crassa glutamine auxotrophs.

This work reports on the isolation and characterization of two glutamine auxotrophs in Neurospora crassa. The mutations responsible for the glutamine-requiring phenotype were very closely linked, and one of them proved to be recessive to wild type. The mutations impaired the conversion of glutamic acid to glutamine and resulted in changes of both the activity and oligomeric structure of the enzyme glutamine synthetase.     

Purification and studies of some physicochemical properties of glutamine synthetase of Neurospora crassa.

Glutamine synthetase (EC 6.3.1.2) of Neurospora crassa was purified to near homogeneity by chromatography on a glutamate-Sepharose affinity column. Its properties, including molecular weight, subunit structure, amino acid composition, and approximate alpha-helix content, have been examined. In the native state, this enzyme has been demonstrated by gel filtration to be an octamer of molecular weight 360,000 and as having a sedimetation coefficient of 13.2 S by sedimentation velocity measurements. Circular dichroism spectra in the far ultraviolet range suggest an approximate alpha-helix content of 23-24%. The subunit generated by treatment with urea was found to be 45,000 daltons by gel filtration methods and a molecular weight of 46,000 was calculated for the monomer obtained by sodium dodecyl sulphate (SDS) treatment and electrophoresis in SDS-polyacrylamide gels. Interprotomeric cross-linking experiments, using diimidoesters, suggest the presence of two noncovalently linked tetramers comprising the native octameric structure. Amino acid analyses revealed the presence of six tryptophans, four half cystines, and nine methionine residues per monomer of 45,000 daltons.     

Heterogeneity of glutamine synthetase polypeptides in Neurospora crassa

Purified preparations of Neurospora crassa glutamine synthetase contain two nonidentical polypeptides that can be separated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 7 M urea. These polypeptides are synthesized both in vivo and in a heterologous cell-free protein-synthesizing system. The data presented indicate that both polypeptides contain an active site for glutamine synthetase activity and suggest that there is not a precursor-product relationship between them.     

Omega-amidase pathway in the degradation of glutamine in Neurospora crassa.

Evidence for the participation of the glutamine transaminase-omega-amidase pathway in the utilization of glutamine in Neurospora crassa was obtained. Its participation is indicated by the in vitro activities of glutamine transaminase and omega-amidase, the in vivo accumulation of alpha-ketoglutaramate when an inhibitor of transamidases is present, and the inhibition by aminooxyacetic acid and 6-diazo-5-oxo-L-norleucine of the ammonium excreted in the presence of glutamine by a mutant strain that lacks glutamate dehydrogenase and glutamate synthase.     

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

The effect of the nitrogen and carbon sources in the regulation of g$\text{l}$u tamine synthetase has been studied in fed-batch cultures of $\text{Neurospora crassa}$. The limitation of ammonium in an excess of the carbon source, leads to an accumulation of α-ketoglutarate and elevation of glutamine sy$\text{n}$ thetase. The limitation of sucrose in an excess of ammonium results in a decrease in glutamine synthetase activity. These results indicate that the carbon source exerts a positive control in the regulation of glutamine synthetase.     

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.     

Nitrogen source regulates glutamine synthetase mRNA levels in Neurospora crassa.

Neurospora crassa glutamine synthetase mRNA was measured by its capacity to direct the synthesis of the specific protein in a cell-free system derived from rabbit reticulocytes. N. crassa cultures grown on glutamate as the sole nitrogen source had higher mRNA activities than did those grown on glutamine. The differences were about 10-fold when polysomal RNA was used for translation and about 5-fold when either total cellular RNA or polyadenylic acid-enriched cellular RNA was used. These data indicate that in exponentially growing N. crassa, the nitrogen source regulates glutamine synthetase by adjusting specific mRNA levels.     

Regulation of glutamine synthesis by glycine and serine in Neurospora crassa.

The biosynthetic activities of the polypeptide subunits alpha and beta of glutamine synthetase (GS) were inhibited in vitro by glycine and serine. These amino acids inhibited the growth of a mutant strain with partial GS activity when grown on glutamate as the nitrogen source and also blocked the synthesis of the glutamine in vivo, thus demonstrating the inhibitory effect on GS activity in vivo. Glycine and serine lowered the intracellular glutamine pool and regulated GS beta synthesis. A preferential induction of synthesis of the GS beta polypeptide was observed when either of these amino acids was present in the medium. On this basis, we obtained a glycine-sensitive mutant which showed a structural alteration of the GS beta polypeptide. The double regulatory effect of either glycine or serine on glutamine synthesis may be considered an example of the regulation of glutamine synthesis by alpha-amino nitrogen. It may be a mechanism that regulates the assimilation of ammonium into glutamate versus glutamine.     

Mobilization of vacuolar arginine in Neurospora crassa. Mechanism and role of glutamine

Nitrogen starvation has been shown to increase the cytosolic arginine concentration and to accelerate protein turnover in mycelia of Neurospora crassa. The cytosolic arginine is derived from a metabolically inactive vacuolar pool. Redistribution of arginine between cytosolic and vacuolar compartments is the result of mobilization of this metabolite in response to nitrogen starvation. Mobilization of arginine (and purines) also occurred in response to glutamine limitation, but arginine accumulated upon proline starvation. These observations indicate that mobilization is a consequence of glutamine limitation rather than a general response to amino acid starvation (or limitation). Analysis of the amino acid pools in mycelia subjected to starvation or limitation suggests that glutamine (or a metabolite derived from glutamine) provides a signal which determines the metabolic fate of vacuolar arginine. The results are consistent with the hypothesis that vacuolar compartmentation provides a readily available store of nitrogen-rich compounds to be utilized during differentiation or under conditions of nutritional stress.     

Genetic and biochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs and their revertants.

In this paper we present the isolation and characterization of glutamine auxotrophs of Neurospora crassa and their revertants. The results show that although various enrichment procedures were used, we found only two types of auxotrophs. Genetic crosses performed between the different mutants showed that the mutations responsible for their phenotypes were highly linked and probably affected the same gene. The biochemical characterization of the glutamine synthetase polypeptides of the different mutants showed that both types contained the alpha monomer. However, in place of the normal beta monomer, each type had a new polypeptide differing from normal beta either in its molecular weight or in its isoelectric point. On the other hand, the revertants had only the alpha monomer and were capable of growing without glutamine. On the basis of these data, we propose that the lack of glutamine synthetase activity in the auxotrophs is due to the interaction of the altered beta with the alpha monomer, and as a consequence the alpha monomer of the revertants regains its activity because of the absence of the altered beta.     

Characterization of Neurospora crassa catabolic dehydroquinase purified from N. crassa and Escherichia coli.

1. Neurospora crassa catabolic dehydroquinase has been purified from N. crassa and Escherichia coli. 2. Protein-sequence and gel-electrophoretic data show that apparently pure, homogeneous native dehydroquinase is a mixture of intact and proteinase-cleaved enzyme monomers. 3. Protein-sequence data and steady-state kinetics show that the catabolic dehydroquinase gene of N. crassa is expressed with fidelity in E. coli.     

Transient kinetic studies of Neurospora crassa cytochrome c oxidase.

The reaction of Neurospora crassa cytochrome c oxidase with CO was studied by flash-photolysis and rapid-mixing experiments, leading to the determination of the association and dissociation rate constants (7 X 10(4) M-1 X s-1 and 0.02s-1 respectively). Pre-steady-state kinetic investigations of the catalytic properties of the enzyme showed that under proper conditions Neurospora cytochrome c oxidase can be 'pulsed', i.e. activated, like the mammalian enzyme. The 'pulsed' species is spectroscopically different from the 'resting' one, and the decay into the 'resting' state is fast (t1/2 approx. 3 min).