Assimilatory Nitrite Reductase Research Articles

Purification and characterization of assimilatory nitrite reductase from Candida utilis

Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

Methylammonium-resistant mutants of Nicotiana plumbaginifolia are affected in nitrate transport.

This work reports the isolation and preliminary characterization of Nicotiana plumbaginifolia mutants resistant to methylammonium. Nicotiana plumbaginifolia plants cannot grow on low levels of nitrate in the presence of methylammonium. Methylammonium is not used as a nitrogen source, although it can be efficiently taken up by Nicotiana plumbaginifolia cells and converted into methylglutamine, an analog of glutamine. Glutamine is known to repress the expression of the enzymes that mediate the first two steps in the nitrate assimilatory pathway, nitrate reductase (NR) and nitrite reductase (NiR). Methylammonium has therefore been used, in combination with low concentrations of nitrate, as a selective agent in order to screen for mutants in which the nitrate pathway is de-repressed. Eleven semi-dominant mutants, all belonging to the same complementation group, were identified. The mutant showing the highest resistance to methylammonium was not affected either in the utilization of ammonium, accumulation of methylammonium or in glutamine synthase activity. A series of experiments showed that utilization of nitrite by the wild-type and the mutant was comparable, in the presence or the absence of methylammonium, thus suggesting that the mutation specifically affected nitrate transport or reduction. Although NR mRNA levels were less repressed by methylammonium treatment of the wild-type than the mutant, NR activities of the mutant remained comparable with or without methylammonium, leading to the hypothesis that modified expression of NR is probably not responsible for resistance to methylammonium. Methylammonium inhibited nitrate uptake in the wild-type but had only a limited effect in the mutant. The implications of these results are discussed.

The electrochemical reduction of iron porphyrin nitrosyls in the presence of weak acids

The reduction of coordinated nitrosyls to ammonia in assimilatory nitrite reductases consists of a series of electron transfer/ protonation steps. Very little detail is known about these steps, either from the enzyme itself or model complexes. In this work, the mechanism for the reduction of iron nitrosyl complexes, which are nitrite reductase models, was examined in solutions with limited proton availability. In this way, the reduction mechanism can be investigated in a system which has been shown quantitatively to produce ammonia. The reduction mechanism was investigated for the reduction of Fe(P)(NO), where P is porphyrins, hydroporphyrins or oxoporphyrins, in the presence of substituted phenols using pulse polarography. In THF, the limiting current for the first wave (wave I) was unaffected by the presence of phenol, while the half-wave potential shifted to positive potentials at high concentrations of phenol. The shift in E 1 2 of the first wave, E 1 2 ,I , was consistent with a mechanism which involved the protonation of Fe(TPP)(NO) − with two protons to form Fe(TPP)(NH 2O +). From the variation in the half-wave potential, the equilibrium constant for the formation of Fe(TPP)(NH 2O +) from Fe(TPP)(NO) − was measured for a series of phenols. The chemical and electrochemical reversibility of the first wave in the presence of phenol was verified by the Nernstian shape of the waves, by square-wave voltammetry, and by shifts caused by the presence of phenolate ion. The reduction in the presence of 2,6-dichlorophenol was an exception in that only one proton was transferred. In addition to the changes in the first wave, a new wave was observed between the first and second waves of Fe(TPP)(NO). This new wave corresponded to a three-electron reduction of Fe(TPP)(NO) −. The kinetics of this reduction were monitored using normal pulse polarography. The behavior of the new wave in the presence of phenol was consistent with a CE mechanism which involved two phenol molecules in equilibrium with Fe(TPP)(NO) − to form Fe(TPP)(NH 2O +) prior to the electron transfer. The rate constant for the reduction was measured for all the phenols for which the equilibrium constant for the formation of Fe(TPP)(NH 2O +) could be measured. Changes in the macrocycle from porphyrin to isobacteriochlorin led to a significant increase in the rate of nitrosyl reduction, while the rate decreased substantially for the oxoporphyrins. Most of the changes in the rate of nitrosyl reduction were consistent with changes in the basicity of the Fe(P)(NO) − complex. It is interesting to note that Fe(P)(NH 2O +), the reactive intermediate, was formed most readily for the assimilatory nitrite reductase models (iron isobacteriochlorins), and least readily for the dissimilatory nitrite reductase models (iron dioxo-isobacteriochlorin).

Molecular cloning, characterization, and nucleotide sequence of nit-6, the structural gene for nitrite reductase in Neurospora crassa.

The Neurospora crassa assimilatory nitrite reductase structural gene, nit-6, has been isolated. A cDNA library was constructed from poly(A)+ RNA isolated from Neurospora mycelia in which nitrate assimilation had been induced. This cDNA was ligated into lambda ZAP II (Stratagene) and amplified. This library was then screened with a polyclonal antibody specific for nitrite reductase. A total of six positive clones were identified. Three of the six clones were found to be identical via restriction digests, restriction fragment length polymorphism mapping, Southern hybridization, and some preliminary sequencing. One of these cDNA clones (pNiR-3) was used as a probe in Northern assays and was found to hybridize to a 3.5-kb poly(A)+ RNA whose expression is nitrate inducible and glutamine repressible in wild-type mycelia. pNiR-3 was used to probe an N. crassa genomic DNA library in phage lambda J1, and many positive clones were isolated. When five of these clones were tested for their ability to transform nit-6 mutants, one clone consistently generated many wild-type transformants. The nit-6 gene has been subcloned to generate pnit-6. The nit-6 gene has been sequenced and mapped; its deduced amino acid sequence exhibits considerable levels of homology to the sequences of Aspergillus sp. and Escherichia coli nitrite reductases. Several pnit-6 transformants have been propagated as homokaryons. These strains have been assayed for the presence of multiple copies of the nit-6 gene, as well as nitrite reductase activity.

Active site structure of assimilatory sulfite and nitrite reductases

Active site structure of assimilatory sulfite and nitrite reductases

The Expression of Nitrate and Nitrite Reductases in Free-living Groundnut Rhizobium Strain 6050: Effect of Combined Nitrogen Sources

Summary In groundnut Rhizobium strain 6050, which contains a new pathway of nitrate dissimilation termed as “nitrate - ammonification”, in vivo expression of nitrate reductase activity under microaerobic conditions did not require added nitrate. However, addition of nitrate to the growth medium enhanced the activity of the enzyme. On the contrary, in vivo expression of assimilatory nitrite reductase activity required nitrate or nitrite. Optimum concentration of nitrate for the maximum expression of NR and NiR activities was found to be 8 mM. Ammonia lowered the in vivo activities of both NR and NiR.

Chromosomal location and function of genes affecting Pseudomonas aeruginosa nitrate assimilation.

Seven known genes control Pseudomonas aeruginosa nitrate assimilation. Three of the genes, designated nas, are required for the synthesis of assimilatory nitrate reductase: nasC encodes a structural component of the enzyme; nasA and nasB encode products that participate in the biosynthesis of the molybdenum cofactor of the enzyme. A fourth gene (nis) is required for the synthesis of assimilatory nitrite reductase. The remaining three genes (ntmA, ntmB, and ntmC) control the assimilation of a number of nitrogen sources. The nas genes and two ntm genes have been located on the chromosome and are well separated from the known nar genes which encode synthesis of dissimilatory nitrate reductase. Our data support the previous conclusion that P. aeruginosa has two distinct nitrate reductase systems, one for the assimilation of nitrate and one for its dissimilation.

Purification and properties of the assimilatory nitrite reductase from barley Hordeum vulgare leaves

The assimilatory nitrite reductase (ferredoxin: nitrite oxidoreductase, EC 1.7.7.1) from barley (Hordeum vulgare L.) leaves has been purified over 1500-fold with a recovery of 30% and a specific activity of 84 mumol of nitrite reduced/min per mg of protein. The purification procedure includes (NH4)2SO4 fractionation, ion-exchange and molecular-sieve chromatographies and, finally, ferredoxin-Sepharose-4B affinity chromatography. The enzyme appears homogeneous by polyacrylamide gel electrophoresis and consists of a single polypeptide chain with an Mr of 61 000. The absorption spectrum of the pure enzyme was typical of a haem-containing protein. The enzyme showed low thermostability and was specific for ferredoxin (Km 0.4 microM), although reduced Methyl Viologen (Km 120 microM) was also effective. The same Km value for nitrite (250 microM) was obtained with both electron carriers. Cyanide acted as a powerful pure competitive inhibitor of enzyme with respect to nitrite (Ki 40 microM). Thiol-blocking agents also caused considerable inhibition, but only the ferredoxin-driven activity was significantly inhibited by sulphite and hydroxylamine.

Nitrite reductase fromAchromobacter fischeri

Nitrite reductase from Achromobacter fischeri catalyzes the six electron reduction of nitrite to ammonia [1]. The enzyme which has a relative mass of 80000 is composed of two subunits of equivalent size which are covalently bonded by a disulfide bridge [2]. The enzyme contains two ctype haems/mol but non-haem iron is absent [1]. It is therefore distinct from the assimilatory nitrite reductases which contain sirohaem and iron-sulfur clusters [35]. A number of electron donors are utilized by the Achromobacter enzyme including FADH 2 and F M N H 2 but the Ki n values for these are much higher than for BVH. Reduced ferredoxin from either spinach or Clostridium pasteurianum which serves as an electron donor for assimilatory nitrite reductases [6] is ineffective with the Achromobacter enzyme. In this paper we show that reduced flavodoxin (Fld) from Chondrus crispus, a eukaryotic red alga, is a very effective donor for Achromobacter nitrite reductase with a K m value similar to BVH. Although hydroxylamine serves as an acceptor, its K m value is approximately two orders of magnitude higher than for nitrite. We have attempted to determine whether hydroxylamine is an intermediate during nitrite reduction by reducing

Characterization and Catalytic Properties of Nitrite Reductase from Anabaena sp. 7119

Summary The assimilatory nitrite reductase from the cyanobacterium Anabaena sp. 7119 (formerly designated Nostoc muscorum ) is an adaptive enzyme whose synthesis is nutritionally repressed by the presence of ammonia in the culture medium. The enzyme is a soluble protein and catalyzes the stoichiometric reduction of nitrite to ammonia. In crude extracts, ferredoxin is an efficient electron donor when reduced either photosynthetically with subcellular particles, enzymatically with a NADPH-generating system and NADP reductase, or chemically with dithionite. However, a 763-fold purified nitrite reductase preparation catalyzes the reduction of nitrite with ferredoxin chemically reduced with dithionite, but it is ineffective when reduced either photosynthetically or enzymatically. Hydroxylamine and cyanide are competitive inhibitors with respect to nitrite of the ferredoxin- nitrite reductase (EC. 1.7.7.1.). The enzymatic activity is also inhibited by o-phenanthroline and sulfite, but not by p-hydroxymercuribenzoate.

Purification to homogeneity of spinach nitrite reductase by ferredoxin-sepharose affinity chromatography.

Assimilatory nitrite reductase was purified 1,700-fold with a yield of 22% from spinach leaves with a procedure involving ammonium sulfate fractionation, DEAE-cellulose and DEAE-Sephadex chromatography, gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous as shown by disc and SDS-gel electrophoresis with a specific activity (mumol NO2-reduced/min/mg protein) of 140.

Ferredoxin-sepharose affinity chromatography for the purification of assimilatory nitrite reductase

Ferredoxin-sepharose affinity chromatography for the purification of assimilatory nitrite reductase

A reduced pyridine nucleotides-diaphorase activity associated to the assimilatory nitrite reductase complex from Neurospora crassa.

The Neurospora crassa assimilatory NAD(P)H-nitrite reductase complex has associated a NAD(P)H-diaphorase activity. 1. This NAD(P)H-diaphorase activity can use either mammalian cytochrome c, 2,6--dichlorophenol-indophenol, ferricyanide, or menadione as electron acceptor from the reduced pyridine nucleotides, and requires flavin adenine dinucleotide for maximal activity. 2. It is inhibited by p-hydroxymercuribenzoate, 1 muM, and it is unaffected by cyanide, sulfite, or arsenite at concentrations which completely inhibit the NAD(P)H-nitrite reductase activity. 3. Flavin adenine dinucleotide specifically protects the NAD(P)H-diaphorase activities, but not the NAD(P)H-nitrite reductase activities, against thermal inactivation. 4. In vitro preincubation of the Neurospora crassa nitrite reductase complex with reduced pyridine nucleotides plus flavin adenine dinucleotide inactivates the NAD(P)H-nitrite reductase activities, but does not affect the NAD(P)H-diaphorase activities, indicating that this nitrite reductase inactivation occurs in the part of the enzyme that contain the nitrite reducing center.

Siroheme: a prosthetic group of the Neurospora crassa assimilatory nitrite reductase.

The Neurospora crassa assimilatory nitrite reductase (EC 1.6.6.4) catalyzes the NADPH-dependent reduction of nitrite to ammonia, a 6-electron transfer reaction. Highly purified preparations of this enzyme exhibit absorption spectra which suggest the presence of a heme component (wavelength maxima for oxidized senzyme: 390 and 578 nm). There is a close correspondence between nitrite reductase activity and absorbance at 400 nm when partially purified nitrite reductase preparations are subjected to sucrose gradient centrifugation. In addition, a role for an iron component in the formation of active nitrite reductase is indicated by the fact that nitrate-induced production of nitrite reductase activity in Neurospora mycelia in vivo requires the presence of iron in the induction medium. The heme chromophore present in Neurospora nitrite reductase preparations is reducible by NADPH. Complete reduction, however, requires the presence of added FAD. The NADPH-nitrite reductase activity of the enzyme is also dependent upon addition of FAD. A spectrally unique complex is formed between the heme chromophore and nitrite (or a reduction product thereof) when nitrite is added to NADPH-reducted enzyme. Carbon monoxide forms a complex with the heme chromophore of nitrite reductase with an intense alpha-band maximum at 590 nm and a beta-band of lower intensity at 550 nm. CO is an inhibitor of NADPH-nitrite reductase activity. Spectrophotometrically detectable CO complex formation and Co inhibition of enzyme activity share the following properties...

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.

Purification and Properties of the Neurospora crassa Assimilatory Nitrite Reductase

Abstract The Neurospora crassa (5297a) assimilatory nitrite reductase (EC 1.6.6.4) catalyzes the NADPH-dependent formation of ammonia from nitrite, a 6 electron reduction. The enzyme has been purified 90-fold by ammonium sulfate fractionations, polyethylene glycol treatment (to remove nucleic acids), DEAE-cellulose, and molecular sieve chromatography. Associated with this NADPH-nitrite reductase activity are NADH-nitrite reductase activity, dithionite-nitrite reductase activity, NADPH-hydroxylamine reductase activity, and NADH-hydroxylamine reductase activity. All the activities are manifestations of a single protein complex as shown by their co-elution from DEAE-cellulose and Sephadex G-200 columns, and by their co-sedimentation during sucrose density gradient centrifugation. The molecular weight was calculated to be 290,000 using a relative s0.72520, w value of 9.4 determined by sucrose density gradient centrifugation and a Stokes radius of 76 A estimated from Sephadex G-200 gel filtration data. The absorption spectrum of a partially purified sample of nitrite reductase exhibited maxima at 405, 555, 585, and g600 nm showing some characteristics of other nitrite and sulfite reducing enzymes which have been studied. The apparent Km for nitrite is 0.01 mm whereas that for hydroxylamine is 2 orders of magnitude higher, 3 mm, suggesting that nitrite is the physiological substrate. The reduced pyridine nucleotide-dependent activities, which require FAD for maximal activity, are more heat-labile and more sensitive to inhibition by p-hydroxymercuribenzoate than the dithionite-dependent activity. All the activities of the nitrite reductase complex are sensitive to inhibition by cyanide, sulfide, sulfite, and o-phenanthroline. The enzyme activities are inhibited on preincubation with either reduced pyridine nucleotide electron donor (NADPH or NADH), provided FAD is present. The electron acceptors, nitrite and hydroxylamine, protect against this inactivation. No evidence of feedback inhibition was found with the variety of nitrogenous compounds tested.

Reduced nicotinamide-adenine dinucleotide-nitrite reductase from Azotobacter chroococcum.

1. The assimilatory nitrite reductase of the N(2)-fixing bacterium Azotobacter chroococcum was prepared in a soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties have been studied. 2. The enzyme is a FAD-dependent metalloprotein (mol.wt. about 67000), which stoicheiometrically catalyses the direct reduction of nitrite to NH(3) with NADH as the electron donor. 3. NADH-nitrite reductase can exist in two either active or inactive interconvertible forms. Inactivation in vitro can be achieved by preincubation with NADH. Nitrite can specifically protect the enzyme against this inactivation and reverse the process once it has occurred. 4. A. chroococcum nitrite reductase is an adaptive enzyme whose formation depends on the presence of either nitrate or nitrite in the nutrient solution. 5. Tungstate inhibits growth of the microorganism very efficiently, by competition with molybdate, when nitrate is the nitrogen source, but does not interfere when nitrite or NH(3) is substituted for nitrate. The addition of tungstate to the culture media results in the loss of nitrate reductase activity but does not affect nitrite reductase.