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Nitrite Reductase (nitrite + reductase)
Selected AbstractsImplication of the glutamine synthetase/glutamate synthase pathway in conditioning the amino acid metabolism in bundle sheath and mesophyll cells of maize leavesFEBS JOURNAL, Issue 12 2008Marie-Hélène Valadier We investigated the role of glutamine synthetases (cytosolic GS1 and chloroplast GS2) and glutamate synthases (ferredoxin-GOGAT and NADH-GOGAT) in the inorganic nitrogen assimilation and reassimilation into amino acids between bundle sheath cells and mesophyll cells for the remobilization of amino acids during the early phase of grain filling in Zea mays L. The plants responded to a light/dark cycle at the level of nitrate, ammonium and amino acids in the second leaf, upward from the primary ear, which acted as the source organ. The assimilation of ammonium issued from distinct pathways and amino acid synthesis were evaluated from the diurnal rhythms of the transcripts and the encoded enzyme activities of nitrate reductase, nitrite reductase, GS1, GS2, ferredoxin-GOGAT, NADH-GOGAT, NADH-glutamate dehydrogenase and asparagine synthetase. We discerned the specific role of the isoproteins of ferredoxin and ferredoxin:NADP+ oxidoreductase in providing ferredoxin-GOGAT with photoreduced or enzymatically reduced ferredoxin as the electron donor. The spatial distribution of ferredoxin-GOGAT supported its role in the nitrogen (re)assimilation and reallocation in bundle sheath cells and mesophyll cells of the source leaf. The diurnal nitrogen recycling within the plants took place via the specific amino acids in the phloem and xylem exudates. Taken together, we conclude that the GS1/ferredoxin-GOGAT cycle is the main pathway of inorganic nitrogen assimilation and recycling into glutamine and glutamate, and preconditions amino acid interconversion and remobilization. [source] Copper-containing nitrite reductase from Pseudomonas chlororaphis DSM 50135FEBS JOURNAL, Issue 12 2004Evidence for modulation of the rate of intramolecular electron transfer through nitrite binding to the type 2 copper center The nitrite reductase (Nir) isolated from Pseudomonas chlororaphis DSM 50135 is a blue enzyme, with type 1 and type 2 copper centers, as in all copper-containing Nirs described so far. For the first time, a direct determination of the reduction potentials of both copper centers in a Cu-Nir was performed: type 2 copper (T2Cu), 172 mV and type 1 copper (T1Cu), 298 mV at pH 7.6. Although the obtained values seem to be inconsistent with the established electron-transfer mechanism, EPR data indicate that the binding of nitrite to the T2Cu center increases its potential, favoring the electron-transfer process. Analysis of the EPR spectrum of the turnover form of the enzyme also suggests that the electron-transfer process between T1Cu and T2Cu is the fastest of the three redox processes involved in the catalysis: (a) reduction of T1Cu; (b) oxidation of T1Cu by T2Cu; and (c) reoxidation of T2Cu by NO2,. Electrochemical experiments show that azurin from the same organism can donate electrons to this enzyme. [source] Silver (Ag+) reduces denitrification and induces enrichment of novel nirK genotypes in soilFEMS MICROBIOLOGY LETTERS, Issue 2 2007Ingela Noredal Throbäck Abstract The use of silver ions in industry to prevent microbial growth is increasing and silver is a new and an overlooked heavy-metal contaminant in sewage sludge-amended soil. The denitrifying community was the model used to assess the dose-dependent effects of silver ions on microorganisms overtime in soil microcosms. Silver caused a sigmoid dose-dependent reduction in denitrification activity, and no recovery was observed during 90 days. Dentrifiers with nirK, which encodes the copper nitrite reductase, were targeted to estimate abundance and community composition for some of the concentrations. The nirK copy number decreased by the highest addition (100 mg Ag kg,1 soil), but the nirK diversity increased. Treatment-specific sequences not clustering with any deposited nirK sequences were found, indicating that silver induces enrichment of novel nirK denitrifiers. [source] Effects of non-steady-state iron limitation on nitrogen assimilatory enzymes in the marine diatom thalassiosira weissflogii (BACILLARIOPHYCEAE)JOURNAL OF PHYCOLOGY, Issue 1 2000Allen J. Milligan Since the recognition of iron-limited high nitrate (or nutrient) low chlorophyll (HNLC) regions of the ocean, low iron availability has been hypothesized to limit the assimilation of nitrate by diatoms. To determine the influence of non-steady-state iron availability on nitrogen assimilatory enzymes, cultures of Thalassiosira weissflogii (Grunow) Fryxell et Hasle were grown under iron-limited and iron-replete conditions using artificial seawater medium. Iron-limited cultures suffered from decreased efficiency of PSII as indicated by the DCMU-induced variable fluorescence signal (Fv/Fm). Under iron-replete conditions, in vitro nitrate reductase (NR) activity was rate limiting to nitrogen assimilation and in vitro nitrite reductase (NiR) activity was 50-fold higher. Under iron limitation, cultures excreted up to 100 fmol NO2,·cell,1·d,1 (about 10% of incorporated N) and NiR activities declined by 50-fold while internal NO2, pools remained relatively constant. Activities of both NR and NiR remained in excess of nitrogen incorporation rates throughout iron-limited growth. One possible explanation is that the supply of photosynthetically derived reductant to NiR may be responsible for the limitation of nitrogen assimilation at the NO2, reduction step. Urease activity showed no response to iron limitation. Carbon:nitrogen ratios were equivalent in both iron conditions, indicating that, relative to carbon, nitrogen was assimilated at similar rates whether iron was limiting growth or not. We hypothesize that, diatoms in HNLC regions are not deficient in their ability to assimilate nitrate when they are iron limited. Rather, it appears that diatoms are limited in their ability to process photons within the photosynthetic electron transport chain which results in nitrite reduction becoming the rate-limiting step in nitrogenassimilation. [source] On-line optical and X-ray spectroscopies with crystallography: an integrated approach for determining metalloprotein structures in functionally well defined statesJOURNAL OF SYNCHROTRON RADIATION, Issue 5 2008Mark J. Ellis X-ray-induced redox changes can lead to incorrect assignments of the functional states of metals in metalloprotein crystals. The need for on-line monitoring of the status of metal ions (and other chromophores) during protein crystallography experiments is of growing importance with the use of intense synchrotron X-ray beams. Significant efforts are therefore being made worldwide to combine different spectroscopies in parallel with X-ray crystallographic data collection. Here the implementation and utilization of optical and X-ray absorption spectroscopies on the modern macromolecular crystallography (MX) beamline 10, at the SRS, Daresbury Laboratory, is described. This beamline is equipped with a dedicated monolithic energy-dispersive X-ray fluorescence detector, allowing X-ray absorption spectroscopy (XAS) measurements to be made in situ on the same crystal used to record the diffraction data. In addition, an optical microspectrophotometer has been incorporated on the beamline, thus facilitating combined MX, XAS and optical spectroscopic measurements. By uniting these techniques it is also possible to monitor the status of optically active and optically silent metal centres present in a crystal at the same time. This unique capability has been applied to observe the results of crystallographic data collection on crystals of nitrite reductase from Alcaligenes xylosoxidans, which contains both type-1 and type-2 Cu centres. It is found that the type-1 Cu centre photoreduces quickly, resulting in the loss of the 595,nm peak in the optical spectrum, while the type-2 Cu centre remains in the oxidized state over a much longer time period, for which independent confirmation is provided by XAS data as this centre has an optical spectrum which is barely detectable using microspectrophotometry. This example clearly demonstrates the importance of using two on-line methods, spectroscopy and XAS, for identifying well defined redox states of metalloproteins during crystallographic data collection. [source] Evidence for post-translational regulation of NrtA, the Aspergillus nidulans high-affinity nitrate transporterNEW PHYTOLOGIST, Issue 4 2007Ye Wang Summary ,,Here, influx and efflux of , and net fluxes of and , were measured in Aspergillus nidulans mutants niaD171 and niiA5, devoid of nitrate reductase (NR) and nitrite reductase (NiR) activities, respectively. ,,Transcript and protein abundances of NrtA, the A. nidulans principal high-affinity transporter, were determined using semiquantitative reverse transcription-polymerase chain reaction and western blots, respectively. , influx in niaD171 was negligible relative to wild-type values, whereas efflux to influx ratios increased nine-fold. Nevertheless, NrtA mRNA and NrtA protein were expressed at levels more than two-fold and three-fold higher, respectively, in niaD171 than in the wild-type strain. ,,This is the first demonstration of diminished high-affinity influx associated with elevated transporter levels, providing evidence that, in addition to transcriptional regulation, control of NrtA expression operates at the post-translational level. This mechanism allows for rapid control of transport at the protein level, reduces the extent of futile cycling of that would otherwise represent a significant energy drain when influx exceeds the capacity for assimilation or storage, and may be responsible for the rapid switching between the on and off state that is associated with simultaneous provision of to mycelia absorbing . [source] Nitrogen-assimilating enzymes in land plants and algae: phylogenic and physiological perspectivesPHYSIOLOGIA PLANTARUM, Issue 1 2002Ritsuko Inokuchi An important biochemical feature of autotrophs, land plants and algae, is their incorporation of inorganic nitrogen, nitrate and ammonium, into the carbon skeleton. Nitrate and ammonium are converted into glutamine and glutamate to produce organic nitrogen compounds, for example proteins and nucleic acids. Ammonium is not only a preferred nitrogen source but also a key metabolite, situated at the junction between carbon metabolism and nitrogen assimilation, because nitrogen compounds can choose an alternative pathway according to the stages of their growth and environmental conditions. The enzymes involved in the reactions are nitrate reductase (EC 1.6.6.1-2), nitrite reductase (EC 1.7.7.1), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.13-14, 1.4.7.1), glutamate dehydrogenase (EC 1.4.1.2-4), aspartate aminotransferase (EC 2.6.1.1), asparagine synthase (EC 6.3.5.4), and phosphoenolpyruvate carboxylase (EC 4.1.1.31). Many of these enzymes exist in multiple forms in different subcellular compartments within different organs and tissues, and play sometimes overlapping and sometimes distinctive roles. Here, we summarize the biochemical characteristics and the physiological roles of these enzymes. We also analyse the molecular evolution of glutamine synthetase, glutamate synthase and glutamate dehydrogenase, and discuss the evolutionary relationships of these three enzymes. [source] Reciprocal diurnal changes of phosphoenolpyruvate carboxylase expression and cytosolic pyruvate kinase, citrate synthase and NADP-isocitrate dehydrogenase expression regulate organic acid metabolism during nitrate assimilation in tobacco leavesPLANT CELL & ENVIRONMENT, Issue 11 2000W.-R. Scheible ABSTRACT Diurnal changes of transcript levels for key enzymes in nitrate and organic acid metabolism and the accompanying changes of enzyme activities and metabolite levels were investigated in nitrogen-sufficient wild-type tobacco, in transfomants with decreased expression of nitrate reductase, and in nitrate-deficient wild-type tobacco. (i) In nitrogen-sufficient wild-type plants, transcript levels for nitrate reductase (NR, EC 1.6.6.1), nitrite reductase (NIR, EC 1.7.7.1) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were high at the end of the night and decreased markedly during the light period. The levels of these three transcripts were increased and the diurnal changes were damped in genotypes with decreased expression of nitrate reductase. The levels of these transcripts were very low in nitrate-limited wild-type plants, except for a small rise after irrigation with 0·2 mM nitrate. (ii) The levels of the transcripts for cytosolic pyruvate kinase (PK, EC 2.7.1.40), mitochondrial citrate synthase (CS, EC 4.1.3.7) and NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42) were highest at the end of the light period and beginning of the night. These three transcripts increase and the diurnal changes were damped in genotypes with decreased expression of NR. (iii) The diurnal changes of transcript levels were accompanied by changes in the activities of the encoded enzymes. The activities of NR and PEPC were highest in the early part of the light period, whereas the activities of PK and NADP-ICDH were highest later in the light period and during the first part of the night and CS activity was highest at the end of the night. Activity of PEPC, PK, CS and NADP-ICDH increased and the diurnal changes were damped in genotypes with low expression of NR. Activity of all four enzymes decreased in nitrate-limited wild-type plants. (iv) In the light, malate accumulated, citrate decreased, and about 30% of the assimilated nitrate accumulated temporarily as glutamine, ammonium, glycine and serine. These changes were reversed during the night. (v) It is proposed that the diurnal changes of expression facilitate preferential synthesis of malate to act as a counter-anion for pH regulation during the first part of the light period when NR activity is high, and preferential synthesis of 2-oxoglutarate to act as a nitrogen acceptor later in the day when large amounts of nitrogen have accumulated in ammonium, glutamine and other amino acids including glycine in the photorespiration pathway, and NR activity has been decreased. [source] Crystallization and preliminary X-ray diffraction analysis of a complex between the electron-transfer partners hexameric Cu-containing nitrite reductase and pseudoazurinACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Daisuke Hira The complex between Cu-containing nitrite reductase (HdNIR) and its electron-donor protein pseudoazurin (HdPAz) from Hyphomicrobium denitrificans has been crystallized. The crystals were obtained from a mixture of the two proteins using the hanging-drop vapour-diffusion method in the presence of polyethylene glycol (PEG) and 2-methyl-2,4-pentanediol (MPD) as precipitants. SDS,PAGE analysis demonstrated that the crystals contained both proteins. The X-ray diffraction experiment was carried out at SPring-8 and diffraction data were collected to 3.3,Å resolution. The crystals were tetragonal (space group P41212), with unit-cell parameters a = b = 130.39, c = 505.55,Å. Preliminary analysis indicated that there was one HdNIR and at least two HdPAz molecules in the asymmetric unit of the crystal. [source] Expression, purification, crystallization and preliminary X-ray diffraction analysis of the soluble domain of PPA0092, a putative nitrite reductase from Propionibacterium acnesACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Masaki Nojiri The soluble domain (residues 483,913) of PPA0092, a putative copper-containing nitrite reductase from Propionibacterium acnes KPA171202, has been overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 2.4,Å. The crystal belonged to space group P213, with unit-cell parameters a = b = c = 108.63,Å. Preliminary diffraction data show that one molecule is present in the asymmetric unit; this corresponds to a VM of 2.1,Å3,Da,1. [source] Homologues of nitrite reductases in ammonia-oxidizing archaea: diversity and genomic contextENVIRONMENTAL MICROBIOLOGY, Issue 4 2010Rita Bartossek Summary Ammonia-oxidizing archaea are frequent and ubiquitous inhabitants of terrestrial and marine environments. As they have only recently been detected, most aspects of their metabolism are yet unknown. Here we report on the occurrence of genes encoding potential homologues of copper-dependent nitrite reductases (NirK) in ammonia-oxidizing archaea of soils and other environments using metagenomic approaches and PCR amplification. Two pairs of highly overlapping 40 kb genome fragments, each containing nirK genes of archaea, were isolated from a metagenomic soil library. Between 68% and 85% of the open reading frames on these genome fragments had homologues in the genomes of the marine archaeal ammonia oxidizers Nitrosopumilus maritimus and Cenarchaeum symbiosum. Extensions of NirK homologues with C-terminal fused amicyanin domains were deduced from two of the four fosmids indicating structural variation of these multicopper proteins in archaea. Phylogenetic analyses including all major groups of currently known NirK homologues revealed that the deduced protein sequences of marine and soil archaea were separated into two highly divergent lineages that did not contain bacterial homologues. In contrast, another separated lineage contained potential multicopper oxidases of both domains, archaea and bacteria. More nirK gene variants directly amplified by PCR from several environments indicated further diversity of the gene and a widespread occurrence in archaea. Transcription of the potential archaeal nirK in soil was demonstrated at different water contents, but no significant increase in transcript copy number was observed with increased denitrifying activity. [source] Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1MOLECULAR MICROBIOLOGY, Issue 1 2010Melanie Kern Summary Bacterial c -type cytochrome maturation is dependent on a complex enzymic machinery. The key reaction is catalysed by cytochrome c haem lyase (CCHL) that usually forms two thioether bonds to attach haem b to the cysteine residues of a haem c binding motif (HBM) which is, in most cases, a CX2CH sequence. Here, the HBM specificity of three distinct CCHL isoenzymes (NrfI, CcsA1 and CcsA2) from the Epsilonproteobacterium Wolinella succinogenes was investigated using either W. succinogenes or Escherichia coli as host organism. Several reporter c -type cytochromes were employed including cytochrome c nitrite reductases (NrfA) from E. coli and Campylobacter jejuni that differ in their active-site HBMs (CX2CK or CX2CH). W. succinogenes CcsA2 was found to attach haem to standard CX2CH motifs in various cytochromes whereas other HBMs were not recognized. NrfI was able to attach haem c to the active-site CX2CK motif of both W. succinogenes and E. coli NrfA, but not to NrfA from C. jejuni. Different apo-cytochrome variants carrying the CX15CH motif, assumed to be recognized by CcsA1 during maturation of the octahaem cytochrome MccA, were not processed by CcsA1 in either W. succinogenes or E. coli. It is concluded that the dedicated CCHLs NrfI and CcsA1 attach haem to non-standard HBMs only in the presence of further, as yet uncharacterized structural features. Interestingly, it proved impossible to delete the ccsA2 gene from the W. succinogenes genome, a finding that is discussed in the light of the available genomic, proteomic and functional data on W. succinogenes c -type cytochromes. [source] Structure of the M148Q mutant of rusticyanin at 1.5,Å: a model for the copper site of stellacyaninACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2001Michael A. Hough The small blue copper protein rusticyanin from Thiobacillus ferrooxidans contains a type 1 Cu centre with a single axial ligand, Met148, which together with the His-Cys-His trigonal planar ligands produces a distorted trigonal pyramidal coordination geometry to copper. Type 1 Cu sites are found in cupredoxins and several multicopper proteins, including oxidases and nitrite reductases. The role of the axial ligand has been extensively debated in terms of its function in the fine tuning of the redox potential and spectroscopic properties of type 1 Cu sites. Numerous mutations of the Met ligand in azurins have been studied, but interpretation of the results has been complicated by the presence of the additional carbonyl oxygen ligand from Gly45, a neighbouring residue to the coordinating His46. The importance of the axial ligand has been further emphasized by the finding that the type 1 centre in Rhus vernicifera stellacyanin, with the lowest redox potential in a type 1 Cu site of 184,mV, has Gln as the axial ligand, whilst fungal laccase and ceruloplasmin, which have redox potentials of 550,800,mV, have a Leu in this position. Here, the crystal structure of the M148Q mutant of rusticyanin at 1.5,Å resolution is presented. This is a significantly higher resolution than that of the structures of native rusticyanin. In addition, the M148Q structure is that of the oxidized protein while the native structures to date are of the reduced protein. The mutant protein crystallizes with two molecules per asymmetric unit, in contrast to the one present in the native crystal form. This mutant's redox potential (550,mV at pH 3.2) is lowered compared with that of the native protein (,670,mV at pH 3.2) by about 120,mV. The type 1 Cu site of M148Q closely mimics the structural characteristics of the equivalent site in non-glycosylated cucumber stellacyanin (redox potential ,260,mV) and, owing to the absence in rusticyanin of the fifth, carbonyl ligand present in azurin, may provide a better model for the R. vernicifera stellacyanin (redox potential ,184,mV) type 1 Cu site, which also lacks the fifth ligand. Furthermore, the presence of two molecules in the asymmetric unit cell indicates a potential binding region of the redox partners. [source] Nitrite reduction: a ubiquitous function from a pre-aerobic pastBIOESSAYS, Issue 8 2009Francesca Cutruzzolà Abstract In eukaryotes, small amounts of nitrite confer cytoprotection against ischemia/reperfusion-related tissue damage in vivo, possibly via reduction to nitric oxide (NO) and inhibition of mitochondrial function. Several hemeproteins are involved in this protective mechanism, starting with deoxyhemoglobin, which is capable of reducing nitrite. In facultative aerobic bacteria, such as Pseudomonas aeruginosa, nitrite is reduced to NO by specialized heme-containing enzymes called cd1 nitrite reductases. The details of their catalytic mechanism are summarized below, together with a hypothesis on the biological role of the unusual d1 -heme, which, in the reduced state, shows unique properties (very high affinity for nitrite and exceptionally fast dissociation of NO). Our results support the idea that the nitrite-based reactions of contemporary eukaryotes are a vestige of earlier bacterial biochemical pathways. The evidence that nitrite reductase activities of enzymes with different cellular roles and biochemical features still exist today highlights the importance of nitrite in cellular homeostasis. [source] | |||||||||||||