Reginald H. Garrett

Nitrate Assimilation in Fungi

Publisher Summary The chapter analyzes the results available in the process of nitrate assimilation as it occurs in two filamentous ascomycetes— Aspergillus nidulans and Neurospora crassa (N. crassa). Not only are these results in general representative of observations made on other fungi and yeasts, but they provide for the most concise and comprehensive description of nitrate assimilation and its regulation. It is a vagary of scientific fortune that the genetics of nitrate assimilation is best understood for A. nidulans, whereas the enzymology of nitrate assimilation is better characterized in N. crassa , the organism more often subjected to biochemical investigation. The assimilatory reduction of nitrate to ammonia is achieved by a metabolic pathway composed of just two enzymes—namely, nitrate reductase and nitrite reductase, which act in this sequence. Recently both have been purified to homogeneity and detailed characterizations of each are being made. Regulation of nitrate assimilation in fungi is eminently simple and straightforward. Ammonia, the end product of the nitrate assimilatory pathway, strongly represses its expression. This phenomenon is the dominant aspect in regulation of nitrate assimilation.

Nitrate Assimilation in Eukaryotic Cells

Publisher Summary This chapter presents an overview of nitrate assimilation in eukaryotic cells. Nitrate is the predominant form of combined nitrogen available within oxidative environment. Its assimilation is achieved through its biological reduction to ammonium. The subsequent utilization of ammonium to form amino and amido-N compounds provides the link among the plethora of pathways of organic nitrogen metabolism and the pathways of inorganic nitrogen assimilation. The capability to assimilate nitrate is possessed by certain bacteria, some fungi, and virtually all algae and higher plants. It is absent from the animal kingdom. Nitrate assimilation represents substantial energy expenditure by the cell when compared with ammonium utilization because eight reducing equivalents are consumed in the reduction of nitrate to ammonium. Consequently, cells that assimilate nitrate, regulate this pathway to avoid wasteful use of reducing power when the product, ammonium, is available. The form of regulation adopted varies in accordance with the metabolic pattern and status of the cell type but the fundamental purpose of the regulation is the same: to affect an economy of existence. The chapter also explains genetic regulation of nitrate assimilation in fungi.

The induction of nitrite reductase in Neurospora crassa.

Abstract Like nitrate reductase, nitrite reductase in Neurospora crassa is induced by either nitrate of nitrite and is repressed by ammonia. None of the enzymatic activities associated with the nitrite reductase, i.e. NADPH-nitrite reductase, reduced benzyl viologen-nitrite reductase or NADPH-hydroxylamine reductase, were derepressible when ammonia-grown N. crassa mycelia were exposed to media lacking any nitrogen source. After a rapid increase in activity in mycelia during the first 6 h of exposure to nitrate, nitrate reductase exhibits a similarly rapid decrease in activity over the ensuing 6 h of exposure. Nitrite reductase shows the same initial increase in activity. However, in contrast to nitrate reductase, nitrite reductase levels remain constant for an extended period, provided sufficient inducer is present. These observations are considererd in light of possible mechanisms which might govern the expression of the nitrate assimilatory pathway.

The regulation of nitrate assimilation in Neurospora crassa: Biochemical analysis of the nmr-1 mutants

SummaryNeurospora crassa nmr-1 mutants, selected on the basis of their sensitivity to chlorate in the presence of glutamine, have elevated levels of the nitrate assimilation enzymes, NADPH-nitrate reductase and NAD(P)H-nitrite reductase. Immunoelectrophoretic determinations show that the higher nitrate reductase activities in nmr-1 mutants are due to greater enzyme concentrations. The half-life of nitrate reductase in these mutants is unaltered. As in wild-type, expression of nitrate assimilation in nmr-1 mutants is dependent on induction by nitrate. Reduced nitrogen metabolites like ammonium and glutamine still repress this expression in nmr-1 mutants, but not as effectively as in wild-type. Enzymatic activity measurements in double mutant strains confirm that the nit regulatory loci, nit-2 and nit-4/5, are epistatic to nmr-1, but nmr-1 is epistatic to nit-3, the nitrate reductase structural gene. The results imply that nmr-1 is involved in post-transcriptional control of nitrate assimilation.

The role of glutamine synthetase and glutamine metabolism in nitrogen metabolite repression, a regulatory phenomenon in the lower eukaryote Neurospora crassa

SummaryGrowth of Neurospora crassa on media containing NH4+leads to the repression of a variety of permeases and alternative pathways which would generate NH4+, so called “ammonium repression.” The mutant am2 which lacks NADP-GDH is not subject to ammonium repression of nitrate reductase or urea permease, but like the wild type has repressed levels of these systems when grown in the presence of proline, glutamate or glutamine. The glutamine synthetase (GS) mutant gln-la has derepressed levels of the aforementioned systems unless grown with glutamine.The oligomeric state of GS depends upon the nitrogen sufficiency of the cell, a tetrameric form predominates under conditions of nitrogen limitation and an octameric form under conditions of nitrogen sufficiency. We have found that the tetrameric form GS predominates in the mutants am2 and gln-la when they are ammonium derepressed.The mechanism of NH4+repression in N. crassa is thought to entail a cessation of positive gene action by the product of the nit-2 regulatory gene. We propose that under conditions of NH4+sufficiency, and hence glutamine sufficiency, the octameric form of GS represses nit-2 gene expression and thereby achieves ammonium repression.

Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: Evidence for autogenous control by nitrate reductase

SummaryA biochemical analysis of mutants altered for nitrate assimilation in Neurospora crassa is described. Mutant alleles at each of the nine nit (nitrate-nonutilizing) loci were assayed for nitrate reductase activity, for three partial activities of nitrate reductase, and for nitrite reductase activity. In each case, the enzyme deficiency was consistent with data obtained from growth tests and complementation tests in previous studies. The mutant strains at these nit loci were also examined for altered regulation of enzyme synthesis. Such exeriments revealed that mutations which affect the structural integrity of the native nitrate reductase molecule can result in constitutive synthesis of this enzyme protein and of nitrite reductase. These results provide very strong evidence that, as in Aspergillus nidulans, nitrate reductase autogenously regulates the pathway of nitrate assimilation. However, only mutants at the nit-2 locus affect the regulation of this pathway by nitrogen metabolite repression.

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs.

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.

Immunoelectrophoretic determination of nitrate reductase in Neurospora crassa

Abstract Highly selective and sensitive methods for determining nitrate reductase levels independent of any expression of its enzymatic activity have been developed, based on immunoelectrophoretic procedures. Specific antisera raised against Neurospora crassa NADPH-nitrate reductase inhibited the overall NADPH-nitrate reductase activity of this enzyme as well as partial activities of the electron transfer process associated with the nitrate reductase reaction. Three immunological techniques were employed to detect the presence of nitrate reductase-related protein in various Neurospora extracts: (i) a protection against inhibition assay, (ii) rocket immunoelectrophoresis, and (iii) crossed immunoelectrophoresis. These three methods were then applied to an analysis of nitrate reductase gene expression in the nit mutants of N. crassa .

The inhibition of the Neurospora crassa nitrate reductase complex by metal-binding agents

Abstract 1. 1.|The Neurospora crassa nitrate reductase complex is sensitive to inhibition by the metal-binding agents cyanide, sulfide and thiourea. The degree of sensitivity and the inhibitory pattern produced by these inhibitors is dependent on the oxidation state of the enzyme. When oxidized, the nitrate reductase is less sensitive to the inhibitors, and their action is competitive with respect to nitrate. However, if the enzyme is reduced by preincubation with NADPH and FAD, its sensitivity to these particular metal-binding agents is increased and nitrate can no longer competitively alleviate their action. 2. 2.|Artificial electron acceptors for the diaphorase activity of the nitrate reductase complex can, in varying degrees, effect a reversal of this non-competitive inhibition of the reduced enzyme. 3. 3.|Presumably, the molybdenum moiety of the nitrate reductase complex has a greater affinity for the metal-binding agents when it is reduced. The phenomenon could serve to regulate the activity of the nitrate reductase complex.

Studies on the in vitro inactivation of the Neurospora crassa assimilatory nitrite reductase in the presence of reduced pyridine nucleotides plus flavin

In vitro inactivation of Neurospora crassa nitrite reductase (NAD(P)H: nitrite oxidoreductase, EC 1.6.6.4) can be obtained by preincubation of the enzyme with reduced pyridine nucleotide plus FAD. The presence of nitrite or hydroxylamine, electron acceptors for the N. crassa nitrite reductase, or cyanide, sulfite or arsenite, competitive inhibitors with respect to nitrite of this enzyme, protects the enzyme against this inactivation. Anaerobic experiments reveal that oxygen is required in order to obtain complete inactivation of nitrite reductase by preincubation with reduced pyridine nucleotide plus FAD. Also, inactivation is prevented if catalase is included in the preincubation mixture. The presence of hydrogen peroxide in the preincubation mixture increases the sensitivity of nitrite reductase to the in vitro FAD-dependent NAD(P)H inactivation. Neither electron acceptors, competitive inhibitors nor catalase, agents which protect the enzyme against the FAD-dependent NAD(P)H inactivation, can reverse this process once it has occurred.