Induction and Repression of Nitrate Reductase in Neurospora crassa

Synthesis of wild-type Neurospora crassa assimilatory nitrate reductase is induced in the presence of nitrate ions and repressed in the presence of ammonium ions. Effects of several Neurospora mutations on the regulation of this enzyme are shown: (i) the mutants, nit-1 and nit-3, involving separate lesions, lack reduced nicotinamide adenine dinucleotide (NADPH)-nitrate reductase activity and at least one of three other activities associated with the wild-type enzyme. The two mutants do not require the presence of nitrate for induction of their aberrant nitrate reductases and are constitutive for their component nitrate reductase activities in the absence of ammonium ions. (ii) An analog of the wild-type enzyme (similar to the nit-1 enzyme) is formed when wild type is grown in a medium in which molybdenum has been replaced by vanadium or tungsten; the resulting enzyme lacks NADPH-nitrate reductase activity. Unlike nit-1, wild type produced this analog only in the presence of nitrate. Contaminating nitrate does not appear to be responsible for the observed mutants' activities. Nitrate reductase is proposed to be autoregulated. (iii) Mutants (am) lacking NADPH-dependent glutamate dehydrogenase activity partially escape ammonium repression of nitrate reductase. The presence of nitrate is required for the enzyme's induction. (iv) A double mutant, nit-1 am-2, proved to be an ideal test system to study the repressive effects of nitrogen-containing metabolites on the induction of nitrate reductase activity. The double mutant does not require nitrate for induction of nitrate reductase, and synthesis of the enzyme is not repressed by the presence of high concentrations of ammonium ions. It is, however, repressed by the presence of any one of six amino acids. Nitrogen metabolites (other than ammonium) appear to be responsible for the mediation of "ammonium repression."     

Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein

Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose. The molecular weight was estimated by gel filtration to be approximately 200,000. A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000. The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm. A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase. The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor. Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes. Some kinetic properties were also determined.     

Expression of a fully functional cd1 nitrite reductase from Pseudomonas aeruginosa in Pseudomonas stutzeri

Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process. We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions. In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen. After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen. The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1). A purification procedure for the recombinant PaNir has been developed, based on the P. aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor. Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode. The full recovery of the denitrification properties in the P. stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration. Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.     

Purification and characterization of the respiratory nitrate reductase of Bacillus licheniformis.

1. Respiratory nitrate reductase of Bacillus licheniformis was extracted from the bacterial membranes by treatment with deoxycholate and purified to a homogeneous state by means of gel chromatography and anion-exchange chromatography. 2. The enzyme (Mr = 193,000, s20, w = 8.6) consists of two subunits, having apparent molecular weight of 150,000 (alpha subunit) and 57,000 (beta subunit), which are present in an equimolar ratio. It does not contain carbohydrate. Ageing of the enzyme appears to result in splitting of the polypeptide chains at specific sites followed by dissociation and reassociation of the digestion products in various combinations. 3. In contrast to Klebsiella aerogenes repiratory nitrate reductase, which is isolated in a tetrameric form that can be reversibly dissociated into a monomeric form by detergents, B. licheniformis nitrate reductase, after isolation, is always present in a monomeric form. This property is related to the difference in membrane localization of the enzyme in the two organisms. 4. B licheniformis nitrate reductase contains 6.9 atoms of non-heme iron, 6.7 atoms of acid-labile sulfide and 0.93 atoms of molybdenum per molecule of enzyme. The molybdenum seems to be part of a low-molecular weight peptide Mo-cofactor) to which it may be bound by interaction with thiol-groups. 5. Antiserum against the native enzyme contains antibodies against both subunits as well as the Mo-cofactor. The Mo-cofactor does not have any antigenic determininants in common with either the alpha or the beta subunit. Also neither subunit cross-reacts with antiserum against the other subunit. Whereas the respiratory nitrate reductases from K. aerogenes and Escherichia coli are immunologically related, the native enzyme from B. licheniformis does not show any cross-reaction with antiserum prepared against either the K. aerogenes or the E. coli enzyme.     

Cytochrome b from Escherichia coli nitrate reductase. Its properties and association with the enzyme complex

Membrane-bound nitrate reductase purified from Escherichia coli was resolved into two separate forms. The majority of the enzyme complex had a subunit composition of 2A:2B:4C, exhibited cytochrome b spectra, and was found to be stable after purification. A second form of nitrate reductase activity was a modified complex with a subunit composition of 2A:2B and lacked cytochrome. The subunit B from this complex was altered in its mobility on sodium dodecyl sulfate-polyacrylamide gels. The cytochrome-containing enzyme had 28 +/- 2 atoms of iron and 1.35 atoms of molybdenum whereas iron and molybdenum in cytochromeless enzyme were 24 +/- 2 atoms and 1.18 atoms/molecule, respectively. Besides cytochrome-containing nitrate reductase, two other cytochrome b-containing fractions were also resolved. These were cytochrome b associated with formate dehydrogenase and a novel cytochrome b with reduced absorption maxima at 430, 529.5, and 560 nm. Nitrate reductase cytochrome b (subunit C) was isolated from subunits A and B as a partially denatured form and its renaturation was accomplished by dialyzing against hemin. The renatured cytochrome yielded absorption spectra similar to the holoenzyme. The pure cytochrome aggregated upon heating, even in the presence of sodium dodecyl sulfate. It had a high isoelectric point (pH greater than 9.5) and had 45% hydrophobic amino acids.     

The 4784V missense mutation of the dnaA5 and dnaA46 alleles confers a defect in ATP binding and thermoiability in initiation of Escherichia coli DNA replication

The prototrophic bacterium Rhodobacter sphaeroides DSM 158 has a periplasmic nitrate reductase which is induced by nitrate and it is not repressed by ammonium or oxygen. In a Tn5 mutant lacking nitrate reductase activity, transposon insertion is localized in a 1.2 kb EcoRI fragment. A 0.6 kb BamHI-EcoRI segment of this region was used as a probe to isolate, from the wild-type strain, a 6.8 kb Pstl fragment carrying the putative genes coding for the periplasmic nitrate reductase. In vivo protein expression and DNA sequence analysis reveal the presence in this region of three genes, napABC, probably organized in an operon. These genes are required for nitrate reduction, as deduced by mutational and complementation studies. The napA gene codes for a protein with a high homology to the periplasmic nitrate reductase from Alcali-genes eutrophus and, to a lesser extent, to other prokaryotic nitrate reductases and molybdenum-containing enzymes. The napB gene product has two haem c-binding sites and shows a high homology with the cytochrome c-type subunit of the periplasmic nitrate reductase from A. eutrophus. NAPA and NAPB proteins appear to be translated with signal peptides of 29 and 24 residues, respectively, indicating that mature proteins are located in the periplasm. The napC gene codes for a 25 kDa protein with a transmembrane sequence of 17 hydrophobic residues. NAPC has four haem c-binding sites and is homologous to the membrane-bound c-type cytochromes encoded by Pseudomonas stutzeri nirT and Escherichia coli torC genes. The phenotypes of defined insertion mutants constructed for each gene also indicate that periplasmic nitrate reductase from R. sphaeroides DSM 158 is a dimeric complex of a 90kDa catalytic subunit (NAPA) and a 15kDa cytochrome c (NAPB), which receives electrons from a membrane-anchored tetrahaem protein (NAPC), thus allowing electron flow between membrane and periplasm. This nitrate-reducing system differs from the assimilatory and respiratory bacterial nitrate reductases at the level of cellular localization, regulatory properties, biochemical characteristics and gene organization.     

Pyridine nucleotide specificity of barley nitrate reductase

NADPH nitrate reductase activity in higher plants has been attributed to the presence of NAD(P)H bispecific nitrate reductases and to the presence of phosphatases capable of hydrolyzing NADPH to NADH. To determine which of these conditions exist in barley (Hordeum vulgare L. cv. Steptoe), we characterized the NADH and NADPH nitrate reductase activities in crude and affinity-chromatography-purified enzyme preparations. The pH optima were 7.5 for NADH and 6 to 6.5 for the NADPH nitrate reductase activities. The ratio of NADPH to NADH nitrate reductase activities was much greater in crude extracts than it was in a purified enzyme preparation. However, this difference was eliminated when the NADPH assays were conducted in the presence of lactate dehydrogenase and pyruvate to eliminate NADH competitively. The addition of lactate dehydrogenase and pyruvate to NADPH nitrate reductase assay media eliminated 80 to 95% of the NADPH nitrate reductase activity in crude extracts. These results suggest that a substantial portion of the NADPH nitrate reductase activity in barley crude extracts results from enzyme(s) capable of converting NADPH to NADH. This conversion may be due to a phosphatase, since phosphate and fluoride inhibited NADPH nitrate reductase activity to a greater extent than the NADH activity. The NADPH activity of the purified nitrate reductase appears to be an inherent property of the barley enzyme, because it was not affected by lactate dehydrogenase and pyruvate. Furthermore, inorganic phosphate did not accumulate in the assay media, indicating that NADPH was not converted to NADH. The wild type barley nitrate reductase is a NADH-specific enzyme with a slight capacity to use NADPH.     

Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12.

The anaerobic regulation of the gene encoding pyruvate formate-lyase from Escherichia coli was investigated. Expression of a pfl'-'lacZ protein fusion demonstrated that the gene is subject to a 12-fold anaerobic induction which can be stimulated a further 2-fold by the addition of pyruvate to the growth medium. Construction of a strain deleted for pfl verified that either pyruvate or a metabolite of glycolysis functions as an inducer of pfl gene expression. Complete anaerobic induction required the presence of a functional fnr gene product. However, the dependence was not absolute since a two- to threefold anaerobic induction could still be observed in an fnr mutant. These results could be confirmed immunologically by analyzing the levels of pyruvate formate-lyase protein present in cells grown under various conditions. It was also shown that pfl'-'lacZ expression was partially repressed by nitrate and that this repression was mediated by the narL gene product.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Evidence for a Second Nitrate Reductase Activity That Is Distinct from the Respiratory Enzyme in Salmonella typhimurium

Significant nitrate reductase activity was detected in mutants of Salmonella typhimurium which mapped at or near chlC and which were incapable of growth with nitrate as electron acceptor. The same mutants were sensitive to chlorate and performed sufficient nitrate reduction to permit anaerobic growth with nitrate as the sole nitrogen source in media containing glucose. The mutant nitrate-reducing protein did not migrate with the wild-type nitrate reductase in polyacrylamide electrophoretic gels. Studies of the electrophoretic mobility in gels of different polyacrylamide concentration revealed that the wild-type and mutant nitrate reductases differed significantly in both size and charge. The second enzyme also differed from the wild-type major enzyme in its response to repression by low pH and its lack of response to repression by glucose. The same mutants were found to be derepressed for nitrite reductase and for a cytochrome with a maximal reduced absorbance at 555 nm at 25°C. This cytochrome was not detected in preparations of the wild type grown under the same conditions. Extracts of these mutants contained normal amounts of the b-type cytochromes which, in the wild type, were associated with nitrate reductase and formate dehydrogenase, respectively, although they could not mediate the oxidation of these cytochromes with nitrate. They were capable of oxidizing the derepressed 555-nm peak cytochrome with nitrate. It is suggested that these mutants synthesize a nitrate-reducing enzyme which is distinct from the chlC gene product and which is repressed in the wild type during anaerobic growth with nitrate.     

Enoate reductases of Clostridia. Cloning, sequencing, and expression

The enr genes specifying enoate reductases of Clostridium tyrobutyricum and Clostridium thermoaceticum were cloned and sequenced. Sequence comparison shows that enoate reductases are similar to a family of flavoproteins comprising 2,4-dienoyl-coenzyme A reductase from Escherichia coli and old yellow enzyme from yeast. The C. thermoaceticum enr gene product was expressed in recombinant Escherichia coli cells growing under anaerobic conditions. The recombinant enzyme was purified and characterized.     

Nitrate reductases inEscherichia coli

Escherichia coli expresses two different membrane-bound respiratory nitrate reductases, nitrate reductase A (NRA) and nitrate reductase Z (NRZ). In this review, we compare the genetic control, biochemical properties and regulation of these two closely related enzyme systems. The two enzymes are encoded by distinct operons located within two different loci on theE. coli chromosome. ThenarGHJI operon, encoding nitrate reductaseA, is located in thechlC locus at 27 minutes, along with several functionally related genes:narK, encoding a nitrate/nitrite antiporter, and thenarXL operon, encoding a nitrate-activated, two component regulatory system. ThenarZYWV operon, encoding nitrate reductase Z, is located in thechlZ locus located at 32.5 minutes, a region which includes anarK homologue,narU, but no apparent homologue to thenarXL operon. The two membrane-bound enzymes have similar structures and biochemical properties and are capable of reducing nitrate using normal physiological substrates. The homology of the amino acid sequences of the peptides encoded by the two operons is extremely high but the intergenic regions share no related sequences. The expression of both thenarGHJI operon and thenarK gene are positively regulated by two transacting factors Fnr and NarL-Phosphate, activated respectively by anaerobiosis and nitrate, while thenarZYWV operon and thenarU gene are constitutively expressed. Nitrate reductase A, which accounts for 98% of the nitrate reductase activity when fully induced, is clearly the major respiratory nitrate reductase inE. coli while the physiological role of the constitutively expressed nitrate reductase Z remains to be defined.Abbreviations NR nitrate reductase On leave from Department of Biochemistry and Molecular Biology, The University of Texas Medical school at Houston, Houston, Texas, 77225, USA     

NADH-dependent nitrate reductase from two-row barley roots: Purification, characteristics and comparison with leaf enzyme

NADH-nitrate reductase (EC 1.6.6.1) was purified 800-fold from roots of two-row barley ( Hordeum vulgare L. cv. Daisen-gold) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on TSK-gel (G3000SW). The specific activity of the purified enzyme was 6.2 μmol nitrite produced (mg protein)⁻¹ min⁻¹ at 30°C.
Besides the reduction of nitrate by NADH, the root enzyme, like leaf nitrate reductase, also catalyzed the partial activities NADH-cytochrome c reductase, NADH-ferricyanide reductase, reduced methyl viologen nitrate reductase and FMNH₂-nitrate reductase. Its molecular weight was estimated to be about 200 kDa, which is somewhat smaller than that for the leaf enzyme. A comparison of root and leaf nitrate reductases shows physiologically similar or identical properties with respect to pH optimum, requirements of electron donor, acceptor, and FAD, apparent K_m for nitrate, NADH and FAD, pH tolerance, thermal stability and response to inorganic orthophosphate. Phosphate activated root nitrate reductase at high concentration of nitrate, but was inhibitory at low concentrations, resulting in increases in apparent K_m for nitrate as well as V_max whereas it did not alter the K_m for NADH.     

Purification and characterization of the respiratory nitrate reductase of Bacillus stearothermophilus

The reduced methylviolegen-nitrate reductase of the obligate thermophile, Bacillus stearothermophilus NCA strain 2184 (ATCC 12016), has been purified to electrophoretic homogeneity, 53-fold with a yield of 12,5%. The purification procedure involved solubilization with octyl glucoside, ammonium sulfate precipitation, ion-exchange, and molecular sieve chromatography. The molecular weight of the enzyme was estimated by polyacrylamide gel electrophoresis to be about 210,000. The enzyme possesses two subunits of 150,000 and 44,000 daltons in equimolar ratio, and no cytochrome. There are 6 atoms of nonheme iron and 12 mol of labile sulfide in 1 mol of the purified enzyme. The 44,000-dalton β subunit is the smallest of all the characterized bacterial nitrate reductases and is very close to the size of the β¹ subunit of Escherichia coli. The various β components of other bacterial nitrate reductases are probably derived from this 44,000-dalton subunit.