Neurospora crassa mutants deficient in asparagine synthetase

Neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. Complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. Recombination analysis with strain S1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group V. In vitro assays with a heterokaryon indicated that the mutation was dominant. Thermal instability of cell extracts from temperature-sensitive strains in an in vitro asparagine synthetase assay determined that the mutations were in the structural gene(s) for asparagine synthetase.     

Pyrimidine-specific carbamyl phosphate synthetase in Neurospora crassa

Two carbamyl phosphate synthetases, the first an arginine-synthetic enzyme (CPS(arg)) and the second a pyrimidine-synthetic enzyme (CPS(pyr)), are shown to be present in Neurospora. The two enzymes can be separated on the basis of size and are distinguished by several different properties. Both CPS(pyr) and CPS(arg) have substrate requirements of adenosine triphosphate, HCO(3) (-), and l-glutamine, although NH(4) (+) in high concentration will partially replace glutamine. CPS(pyr) activity can be completely inhibited by 5 x 10(-4) to 10 x 10(-4)m uridine triphosphate (UTP). CPS(pyr) is cold-labile and can be protected against cold inactivation by UTP. The synthesis of CPS(pyr) and aspartate transcarbamylase (ATC), the initial enzymatic steps of the pyrimidine pathway, are co-derepressed by pyrimidine starvation. Mutations affecting CPS(pyr) and ATC all map at the same locus, pyr-3. Three classes of mutants with respect to the two activities were found: CPS(+)ATC(-), CPS(-)ATC(+), and CPS(-)ATC(-). The distribution of these mutants on the genetic map, together with other data, indicate that the two activities are carried by a bifunctional protein.     

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.     

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.     

Carbamyl phosphate synthetase A of Neurospora crassa.

Carbamyl phosphate synthetase A of Neurospora crassa was partially purified from mitochondrial extracts. It is an extremely unstable enzyme (t 1/2 = 45 min at 25 detrees C) made up of two unequal subunits. The native enzyme has a molecular weight of approximately 175,000, and the large subunit has a molecular weight of about 125,000. Both the native enzyme and its large subunit are quite asymmetric, as revealed by slow sedimentation in sucrose gradents (7.3S and 6.6S, respectively). The small subunit has not been identified physically as a separate entity. The denaturation of the native, glutamine-dependent activity is correlated with dissociation of subunits, the larger of which retains a more stable, ammonia-dependent activity. Neither substrates nor any other agents except glycerol or polyethylene glycol appreciably stabilized the glutamine-dependent activity. Kinetic studies showed the native enzyme to have a Km for glutamine of about 0.16 mM, and a Km for NH4Cl of about 16 mM, at the optimal pH, 8.0. The enzyme, using either N donor, has a K+ requirement for activity, for which NH4+ can substitute. The glutamine leads to glutamate reaction, which requires the small subunit, also requires the large subunit and all reaction substrates for optimal activity. Other evidences of subunit interaction are the greater activity of the native enzyme, as opposed to the large subunit, with low concentrations of adenosine 5'-triphosphate-Mg2+, and in the stimulation of the ammonia-dependent activity of the native enzyme by glycine. Curiously, although the enzyme's role in biosynthesis is confined to the arginine pathway, it is completely indifferent to arginine or its precursors as feedback effectors or activators. The enzyme is compared with carbamyl phosphate synthetases of other organisms.     

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.     

Subunit structure of anthranilate synthetase from Neurospora crassa.

Freshly purified preparations of anthranilate synthetase complex from Neurospora crassa appeared to be homogeneous on polyacrylamide disc gels and were composed of two distinct subunits, 94,000 and 70,000 daltons, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Carboxymethylation of the complex or treatment with guanidine hydrochloride and urea before sodium dodecyl sulfate treatment did not alter the subunit pattern. When the purified complex was iodinated with 125I- or methylated with [14C]dimethylsulfate, no labeled components other than the two subunits stained with Coomassie blue were detected after electrophoresis in the presence of sodium dodecyl sulfate. Although some purified preparations were stable, most were unstable upon storage. Analysis of the unstable preparations on nondenaturing and sodium dodecyl sulfate polyacrylamide disc gels revealed that the complex in these preparations was progressively fragmented to smaller components and subunits upon repeated freeze-thaw treatment or prolonged incubation at or above 4 degrees. Distinct fragments were generated ranging in size down to 25,000 daltons, and some fragments retained some of the activities associated with the anthranilate synthetase complex. On the basis of these and earlier studies, we conclude that anthranilate synthetase from Neurospora crassa is composed of two distinct subunits in an alpha2beta2 structure; one subunit is a trifunctional peptide which contains the catalytic sites for the phosphoribosylanthranilate isomerase and indoleglycerol phosphate synthetase reactions, and associates with the second subunit to form glutamine-dependent anthranilate synthetase. The smaller subunits and components previously reported for this complex are apparently due to protease activity present in purified preparations.     

Mode of action of glycogen branching enzyme from Neurospora crassa

Neurospora crassa branching enzyme [EC 2.4.1.18] acted on potato amylopectin or amylose to convert them to highly branched glycogen-type molecules which consisted of unit chains of six glucose units. The enzyme also acted on the amylopectin beta-limit dextrin, indicating that the enzyme acted on internal glucose chains as well as outer chains. By the combined action of N. crassa glycogen synthase [EC 2.4.1.11] and the branching enzyme, a glycogen-type molecule was formed from UDP-glucose. In the presence of primer glycogen, the glucose transfer reaction was accelerated by the addition of branching enzyme. On the other hand, the glucose transfer reaction by glycogen synthase did not occur without primers. When the branching enzyme was added, the glucose transfer occurred after a short time lag. This recovery of the glucose transfer reaction did not occur upon addition of the inactivated branching enzyme. The structure of the product formed by the combined action of the two enzymes was different from that of the intact N. crassa glycogen with respect to the distribution patterns of the unit chains.     

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.     

Unstable S-Adenosylmethionine synthetase in an ethionine-resistant strain of Neurospora crassa.

A pleitropic ethionine-resistant mutant of Neurospora contains an S-adenosylmethionine synthetase that is labile to heat and dialysis but exhibits normal kinetics for methionine and ethionine.     

Purification and characterization of recombinant FAD synthetase from Neurospora crassa

FAD Synthetase (FADS) [EC 2.7.7.2], the second enzyme in flavin cofactor biosynthetic pathway converts FMN to FAD, plays an important role in many redox reactions. Neurospora crassa FADS (NcFADS) was cloned and overexpressed in E. coli cells. Recombinant NcFADS was purified in high yields of ～8 mg per liter of bacterial culture using a single step glutathione sepharose affinity chromatography. SDS-PAGE and MALDI-MS revealed that NcFADS has a molecular mass of ～31 kDa. Enzyme kinetic analysis monitored by reverse phase HPLC demonstrate a specific activity and k_cat of 1356 nmol/min/mg and 0.69sec^?1 respectively. Steady state kinetic analysis of NcFADS exhibited a K_m of NcFADS for FMN is 2.7 μM and for MgATP^?2 is 88.7 μM. Isothermal titration calorimetry experiments showed that the recombinant protein binds to the substrates with apparent K_d of 20.8 μM for FMN and 16.6 μM for MgATP^?2. Biophysical characterization using intrinsic fluorescence suggests that the enzyme is in folded conformation. Far-UV CD data suggest that the backbone of the enzyme is predominantly in a helical conformation. Differential scanning calorimetry data shows that the T_m is 53 °C ± 1. This is the first report on cloning, purification and characterization of FADS from N. crassa. The specific activity of NcFADS is the highest than any of the reported FADS from any other source. The results obtained in this study is expected to pave way for intensive research aimed to understand the molecular basis for the extraordinarily high turnover rate of NcFADS.     

Two forms of a mitochondrial endonuclease in Neurospora crassa.

Mitochondrial nuclease activity in Neurospora crassa occurs in membrane-bound and soluble forms in approximately equal proportions. These activities apparently are due to the same enzyme, which has an approximate molecular weight of 120 000. A portion of the insoluble enzyme appears to be associated with the inner mitochondrial membrane and is resistant to solubilization by detergent treatment as well as by physical disruption methods.     

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.     

Biosynthesis of glycogen in Neurospora crassa. Kinetic mechanism of UDP-glucose: glycogen 4-alpha-glucosyltransferase.

The kinetic mechanism of glycogen synthase [UDP-glucose: glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11], glucose-6-P-dependent form, from Neurospora crassa has been investigated by initial velocity experiments and studies with inhibitors in the presence of sufficient levels of glucose-6-P. The rate equation was different from those of common two-substrate systems because one of the substrates, glycogen, is also a product. The reaction rates were determined by varying the concentration of one of the substrates while keeping that of the other constant. Double-reciprocal plots of initial velocity measurements were linear and showed converging line patterns. UDP was found to act competitively when the substrate UDP-glucose was varied, but noncompetitively when glycogen was varied. On the basis of these results, it is concluded that glycogen synthase, glucose-6-P-dependent form, from N. crassa has a rapid equilibrium random Bi-Bi mechanism. Rate constant and dissociation constants for each step of this mechanism were estimated.