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.     

Regulation and function of glutamate synthase in Neurospora crassa

In Neurospora crassa two enzymes can provide glutamate: the NADPH dependent GDH and the NADH dependent GOGAT. An elevated GOGAT activity was found in Neurospora wild-type under ammonium limitation in contrast to a 4-fold lower activity on excess of a$\text{m}$ monium. Glutamate and glutamine repress this enzyme. On excess of ammonium the GDH-NADPH deficient mutant am-1 grows poorly with an elevated GOGAT activity. A GOGAT less mutant was found. It presented a lag-phase to grow on ammonium. It is concluded that N. crassa glutamate synthase provides glutamate from low am-monium concentrations. The enzyme was purified to homogeneity and shown to be composed of a single type of monomer with a molecular weight above 200,000.     

The use of affinity elution from Blue Dextran Sepharose by yeast tRNA2Val in the complete purification of the cytoplasmic valyl-tRNA synthetase from Euglena gracilis

Two distinct monomers, α and β participate in the structures of diffe$\text{r}$ ent oligomers of $\text{Neurospora crassa}$ glutamine synthetase (EC 6. 3. 1. 2). In ammonium-limited cultures a tetrameric form composed mainly of α monomers was found. In excess of nitrogen an octameric form composed mainly from β monomers is the predominant oligomeric state. The presence of both monomers was observed in intermediate oligomeric forms.     

Characterization of glutamine requiring mutants of Bacillus subtilis

Mutants of $\text{B. subtilis}$ 168 which exhibited an absolute requirement for glutamine have been isolated and characterized. Of the two mutants studied in detail, one had normal levels of glutamine synthetase and sporulated normally, the other had reduced glutamine synthetase and was asporogenic. Both mutants were mapped close to the $\text{thy}$ A region of the chromosome by PBS1 transduction.A study of spontaneous revertants selected for glutamine prototrophy (or the sporulation character in the case of the asporogenic mutant) led to the conclusion that there is a relationship between the glutamine requirement and sporulation. However, the influence of glutamine could not be entirely explained by the catalytic properties of glutamine synthetase.     

The occurrence of glutamate synthase in algae.

The presence of glutamate synthase in the green algae $\text{Chlorella fusca}$ var. $\text{vacuolata}$ has been demonstrated using a whole cell assay as well as cell free extracts. The assay is complicated by the presence of glutamine (amino): α-oxoglutarate transaminase, but this enzyme can be inhibited by amino oxyacetate. The rates of glutamate synthase activity are sufficient to account for the known rates of nitrate assimilation to occur via the glutamine synthetase/glutamate synthase pathway.     

Studies on the binary and ternary complexes formed by a neurospora glutamate dehydrogenase and its substrates

The NADP⁺ specific glutamate dehydrogenase from wild-type $\text{Neurospora crassa}$ forms a stable binary complex with NADPH. This can combine with L-glutamate, α-ketoglutarate or the substrate analogue D-glutamate to form ternary complexes which can be distinguished by their different fluorescence properties. The affinity of the enzyme for NADPH diminishes with increases in pH or ionic strength of the solution. Experimental data obtained using modified glutamate dehydrogenases from mutant strains of $\text{N. crassa}$ suggest that the reduced-coenzyme binding sites observed fluorimetrically are the same as those observed by enzyme kinetics.     

Regulation of glutamate synthase from Bacillussubtilis by Glutamine

Glutamate synthase, an important enzyme in the assimilation of ammonia, was measured in cultures of $\text{Bacillus}\text{subtilis}$ grown with different nitrogen sources. An attempt was made to correlate the specific activity to the intracellular levels of five metabolites of glutamate metabolism: aspartate, glutamate, glutamine, alanine and $\text{NH}{}_4{}^{\mathrm{+}}$. An inverse relationship was found between the activity of glutamate synthase and the pool level of glutamine. We propose that the intracellular concentration of glutamine is an important element in controlling the level of glutamate synthase.     

Nitrogen fixation in cultured cowpea Rhizobia: inhibition and regulation of nitrogenase activity.

Nitrogenase activity in agar cultures of cowpea rhizobia, strain 32H1, was rapidly inhibited by $\text{NH}{}_4{}^{\mathrm{+}}$ but this was relieved by increased O₂ tension. Inhibition was more rapid than that caused by inhibitors of protein synthesis and was not relieved by methionine sulfoximine or methionine sulfone. Under conditions were nitrogenase activity was inhibited by $\text{NH}{}_4{}^{\mathrm{+}}$, glutamine synthetase and glutamate synthase were substantially unaffected. Glutamate dehydrogenase was undetected in either nitrogenase active or $\text{NH}{}_4{}^{\mathrm{+}}$ inhibited cultures. These results indicate that $\text{NH}{}_4{}^{\mathrm{+}}$ inhibition of nitrogenase activity in strain 32H1 is not effected through glutamine synthetase regulation of nitrogenase synthesis.     

Glutatmine synthetase activity in the chick brain during postnatal growth. Effect of MSO

Glutamine synthetase activity was estimated in the chick cerebral hemispheres, optic lobes and cerebellum between the 1st and the 30th day of postnatal growth. Glutamine synthetase activity is higher in the cerebellum than in the cerebral hemispheres and lowest in the optic lobes at 1 day after hatching; at 30 days after hatching, it is the same in the optic lobes and in the cerebellum and lowest in the cerebral hemispheres. The great increase of glutamine synthetase activity between the 1st and the 4th day after hatching corresponds to the appearance of the heterogeneity of the chick brain glutamate metabolism. The glutamine synthetase activity is inhibited by MSO $\text{in vivo}$ at a concentration of 100 mg kg ^?1 at values of 87, 90 and 89 % in cerebral hemispheres, optic lobes and cerebellum of 1, 2 and 4-day-old chicks. The enzyme inhibition is less pronounced $\text{in vitro}$ and reaches values of about 25 and 75 % for 1 and 10 mM MSO concentrations respectively in the three brain areas of the 1 to 4-day-old chick and values slightly lower in the 30-day-old chick brain.     

The role of O2-limitation in control of nitrogenase in continuous cultures of Rhizobrium sp.

Oxygen-limited continuous cultures of the cowpea $\text{Rhizobium}$ sp. strain CB756, had high levels of nitrogenase activity, which were not significantly affected by excess ammonium ions or glutamine. When the growth-restricting O₂-limitation was partially relieved, nitrogenase was repressed and this was accompanied by increased adenylylation of glutamine synthetase. It is suggested that the restricted supply of ATP interferes with adenylylation of glutamine synthetase during O₂-limited growth, thus preventing repression of nitrogenase in the presence of excess ammonium ions.     

Utilization of ammonia for tryptophan synthesis

A strain of $\text{Escherichia coli}$ in which the glutamine amidotransferase function (anthranilate synthetase component II) of anthranilate synthetase has been deleted synthesizes tryptophan using NH₃-dependent anthranilate synthetase component I (AS-I). In NH₃-limited media this strain is a tryptophan auxotroph. Mutants that acquired the capacity to grow in NH₃-limited media were isolated. Growth of mutant strains in NH₃-limited media correlates with increased AS-I activity. Glutamine-dependent AS activity was not found in any of the mutant strains indicating that another glutamine amidotransferase had not been recruited to function with AS-I.     

Detection of two forms of glutamine synthetase in Bacillus brevis (A. G. 4)

A laboratory isolate of $\text{Bacillus}\text{brevis}$ could grow and sporulate on an amino acid, viz., alanine or glutamate or aspartate as single source of carbon and nitrogen. It failed to sporulate if the amino acid was replaced by the corresponding keto acid and ammonium sulphate in the medium, although, normal growth was observed. One of the key enzymes in nitrogen assimilation, the glutamine synthetase, has been purified by DE-52 and affinity column chromatography from both alanine and pyruvate grown cells. The kinetic and other properties of both of these enzymes were studied. The enzyme isolated from alanine grown cells differed significantly from that isolated from pyruvate grown cells (viz.,pH optima, response to Mg⁺⁺ and other effectors). A possible role of glutamine synthetase in the initiation of bacterial sporulation is discussed.     

5' Cap methylation of homologous poly A(+) RNA by a RNA (guanine-7) methyltransferase from Neurospora crassa.

RNA (guanine-7) methyltransferase, partially purified from $\text{N.}\text{crassa}$ mycelia, catalyzed the transfer of the methyl group from S-adenosylmethionine to the 5′ terminus of both $\text{N.}\text{crassa}$ poly A(+) RNA and reovirus unmethylated mRNA. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated poly A(+) RNA from $\text{N.}\text{crassa}$ yielded the “cap” structures m ⁷G(5′)pppAp and m ⁷G(5′)pppGp in a ratio of 2:1 respectively. RNase T₂ digestion of the $\text{in}\text{vitro}$ methylated reovirus mRNA yielded m ⁷G(5′)pppGp exclusively. The absence of mRNA 2′-0-methyltransferase activity in the enzyme preparation is consistent with the absence of 2′-0-methylation in $\text{N.}\text{crassa}$ mRNA [Seidel, B. L. and Somberg, E. W. (1978) Arch. Biochem. Biophys. $\text{187}$, 108–112]. This is the first isolation of an eucaryotic, cellular RNA (guanine-7) methyltransferase that has been shown to methylate homologous substrate.     

Regulation of carbon and nitrogen flow by glutamate synthase in Neurospora crassa

A glycine-resistant Neurospora crassa mutant (am-132;glyr), derived from the am-132 mutant, was isolated and characterized. [am-132 itself has a deletion in the structural gene for NADP-dependent glutamate dehydrogenase (GDH).] This new mutation also conferred resistance to serine and methionine sulphoximine (MS), which are inhibitors of glutamine synthetase (GS). In addition, the mutant obtained grew better on ammonium than the am-132 parental strain. Resistance to glycine was not due to increased synthesis of glutamine by an altered or induced GS, nor to increased glutamate synthesis by induction of the catabolic NAD-dependent GDH, nor to NADH-dependent glutamate synthase (GOGAT), which was as sensitive to inhibitors as the GOGAT from the parental strain. The glycine-resistance mutation lowered but did not abolish the carbon flow; this resulted in a lower content of tricarboxylic acid cycle intermediates. GOGAT activity was inhibited in vitro by several organic acids and methionine sulphone (MSF). The higher growth rate of the glycine-resistant mutant on ammonium or on ammonium plus glycine, serine or MS was explained by an increased capacity of GOGAT to synthesize glutamate in vivo due to a lower content of inhibitory tricarboxylic acid cycle intermediates; the higher glutamate content overcomes the effect of the GS inhibitors and explains the MSF resistance of the mutant.     

ColE1 hybrid plasmids containing Escherichia coli genes involved in the biosynthesis of glutamate and glutamine

The Clarke-Carbon bank of Escherichia coli strains carrying ColE1 hybrid plasmids was screened for complementation of gdh, gltB, and glnA mutations affecting nitrogen metabolism in E. coli. Plasmids which complemented each one of these mutations were isolated. In every case, the plasmids conferred to otherwise mutant cells the capacity to synthesize the corresponding wild-type enzymes: glutamate dehydrogenase, glutamate synthase, and glutamine synthetase (GS), respectively. For three representative plasmids, endonuclease restriction maps were constructed. One of the plasmids, pACR1, which complemented glnA mutations, including the glnA21::Tn5 insertion, was deemed to carry the glnA⁺ allele. GS synthesis by pACR1 $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ heterozygous merodiploids was subjected to repression by growth on 15 mm NH₄⁺ and had a twofold high derepressed level than wild-type (glnA⁺) haploid cells when grown on 0.5 mm NH₄⁺ or on glutamate as only nitrogen sources. The presence of glutamine as sole nitrogen source promoted repressed GS synthesis in the $\text{glnA}{}^{\mathrm{+}}\text{glnA20}$ merodiploids. By contrast, glutamine allowed almost fully derepressed synthesis of GS in glnA⁺ haploid cells.     

Evidence for methionine sulfoximine as a transition-state analog for glutamine synthetase from NMR and EPR data.

Manganese(II)bound at the “tight” metal ion site of unadenylylated glutamine synthetase (E. coli W) has two rapidly exchanging first coordination sphere water molecules. The solvation number was evaluated from a study of the frequency dependence of $\text{1}\text{pT}{}_{\mathrm{<mtext>1p</mtext>}}$, the paramagnetic contribution to the longitudinal relaxation rate of solvent protons. The number of rapidly exchanging water molecules is reduced to one in the presence of saturating L-glutamate and to ～0.2 when L-methionine SR-sulfoximine (MSOX) is present. MSOX is a linear competitive inhibitor (K_I=3μM) of glutamine synthetase when L-Glu is the substrate. The dissociation constant of MSOX measured by following the 18 fold decrease in $\text{1}\text{pT}{}_{\mathrm{1p}}$ (at 48 MHz) is 30μM and is lowered to ～9μM in the presence of ADP. The high affinity of MSOX for the enzyme suggests that this compound mimicks the “transition-state” for the glutamine synthetase reaction. Further evidence for this postulate is found from the dramatic sharpening of the epr spectrum of enzyme-bound Mn(II) in the presence of MSOX and MSOX plus ADP. The intense change in the epr spectrum arises from reduced solvent accessibility to bound Mn(II) and conformational changes produced by binding MSOX and ADP. The suggestion is made from these data that L-Glu and MSOX bind near or directly to the Mn(II) at the “tight” metal ion site in glutamine synthetase isolated from E. coli W.     

Preferential utilization of glutamine for amination of xanthosine 5'-phosphate to guanosine 5'-phosphate by purified enzymes from Escherichia coli

GMP synthetase was purified 180-fold from $\text{E. coli}$ B and 18-fold from the derepressed purine auxotroph, $\text{E. coli}$ B-96. The enzymes from both sources show the same preference for glutamine over ammonia as amino donor. Each is dimeric, consisting of subunits of molecular weight about 60,000. Thus the two are apparently identical. The similarities between GMP synthetase and xanthosine 5′-phosphate aminase of $\text{E. coli}$ B-96 (N. Sakamoto, G.W. Hatfield, and H.S. Moyed, J. Biol. Chem. (1972) $\text{247}$, 5880–5887) in respect to structure, state of derepression, and behavior during purification, lead us to the conclusion that the synthetase and the aminase are a single entity. We observe no loss or separation of glutamine-dependent activity upon purification of GMP synthetase and we suggest that such loss, reported by other workers, results artifactually by inactivation of an intrinsic glutamine-binding site. GMP synthetase appears not to contain a glutamine-binding subunit which is separable from the xanthosine 5′-phosphate-aminating component.     

Detection of sequence heterology by use of the N. Crassa nucleases

We have used the single-strand specific nucleases of $\text{Neurospora crassa}$ to detect sequence divergencies between two similar DNA molecules: restriction endonuc lease $\text{Eco}$RI produced linears from Simian Virus 40 and a variant of human origin, DAR. Enzyme treatment of the heteroduplex DNA resulted in specific cleavage into two fragments of one-third and two-thirds genome length. These two viral DNAs therefore have at least one region of heterology located about 0.35 map units from the $\text{Eco}$RI site. Due to the known specificities of the $\text{N.crassa}$ nucleases, this technique is applicable to detect mutations in RNA or DNA genomes.     

Ammonia Fixation via Glutamine Synthetase and Glutamate Synthase in the CAM Plant Cissus quadrangularis L

Succulent stems of Cissus quadrangularis L. (Vitaceae) contain glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. The CO₂ and water gas exchanges of detached internodes were typical for Crassulacean acid metabolism plants. During three physiological phases, e.g. in the dark, in the early illumination period after stomata closure, and during the late light phase with the stomata wide open, ¹⁵NH₄Cl was injected into the central pith of stem sections. The kinetics of ¹⁵N labeling in glutamate and glutamine suggested that glutamine synthetase was involved in the initial ammonia fixation. In the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, the incorporation of ¹⁵N derived from ¹⁵NH₄Cl was almost completely inhibited. Injections of amido-¹⁵N glutamine demonstrated a potential for ¹⁵N transfer from the amido group of glutamine into glutamate which was suppressed by the glutamate synthase inhibitor, azaserine. The evidence indicates that glutamine synthetase and glutamate synthase could assimilate ammonia and cycle nitrogen during all phases of Crassulacean acid metabolism.     

Crystals of glutamine synthetase from Escherichia coli.

Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are $\text{a = 140 ± 2}\text{A}\text{̊}$, and $\text{c = 148 ± 2}\text{A}\text{̊}$. The Laue symmetry group is $\text{3}\text{̄}$m P3₁.