Nitrate reductase

Nitrate reductases are molybdoenzymes that reduce nitrate (NO
3
) to nitrite (NO
2
). This reaction is critical for the production of protein in most crop plants, as nitrate is the predominant source of nitrogen in fertilized soils.[2]

nitrate reductase
Nitrate reductase
structure of nitrate reductase A from E. coli[1]
Identifiers
EC number1.7.99.4
CAS number37256-45-4
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Gene OntologyAmiGO / QuickGO
Molybdopterin oxidoreductase (nitrate reductase alpha subunit)
Identifiers
SymbolMolybdopterin
PfamPF00384
InterProIPR006656
PROSITEPDOC00392
SCOP1cxs
SUPERFAMILY1cxs
OPM superfamily3
OPM protein1kqf
4Fe-4S dicluster domain
(nitrate reductase beta subunit)
Identifiers
SymbolFer4_11
PfamPF13247
Nitrate reductase gamma subunit
Identifiers
SymbolNitrate_red_gam
PfamPF02665
InterProIPR003816
SCOP1q16
SUPERFAMILY1q16
TCDB5.A.3
OPM superfamily3
OPM protein1q16
Nitrate reductase delta subunit
Identifiers
SymbolNitrate_red_del
PfamPF02613
InterProIPR003765
Nitrate reductase cytochrome c-type subunit (NapB)
Identifiers
SymbolNapB
PfamPF03892
InterProIPR005591
SCOP1jni
SUPERFAMILY1jni
Periplasmic nitrate reductase protein NapE
Identifiers
SymbolNapE
PfamPF06796
InterProIPR010649

Types

Eukaryotic

Eukaryotic nitrate reductases are part of the sulfite oxidase family of molybdoenzymes. They transfer electrons from NADH or NADPH to nitrate.

Prokaryotic

Prokaryotic nitrate reductases belong to the DMSO reductase family of molybdoenzymes and have been classified into three groups, assimilatory nitrate reductases (Nas), respiratory nitrate reductase (Nar), and periplasmic nitrate reductases (Nap). The active site of these enzymes is a Mo ion that is bound to the four thiolate functions of two pterin molecules. The coordination sphere of the Mo is completed by one amino-acid side chain and oxygen and/or sulfur ligands. The exact environment of the Mo ion in certain of these enzymes (oxygen versus sulfur as a sixth molybdenum ligand) is still debated. The Mo is covalently attached to the protein by a cysteine ligand in Nap, and an aspartate in Nar.[3]

Structure

The transmembrane respiratory nitrate reductase (EC) is composed of three subunits; an alpha, a beta and two gamma. It is the second nitrate reductase enzyme which it can substitute for the NRA enzyme in Escherichia coli allowing it to use nitrate as an electron acceptor during anaerobic respiration.[4]

Nitrate reductase gamma subunit resembles cytochrome b and transfers electrons from quinones to the beta subunit.[5]

The nitrate reductase of higher plants is a cytosolic protein. There exists a GPI-anchored variant that is found on the outer face of the plasma membrane. Its exact function is still not clear.[6]

A transmembrane nitrate reductase that can function as a proton pump (similar to the case of anaerobic respiration) has been discovered in a diatom Thalassiosira weissflogii.[7]

Mechanism

Nitrate molecule binds to the active site with the Mo ion in the +6 oxidation state. Electron transfer to the active site occurs only in the proton-electron transfer stage, where the MoV species plays an important role in catalysis. The presence of the sulfur atom in the molybdenum coordination sphere creates a pseudo-dithiolene ligand that protects it from any direct attack from the solvent. Upon the nitrate binding there is a conformational rearrangement of this ring that allows the direct contact of the nitrate with MoVI ion. This rearrangement is stabilized by the conserved methionines Met141 and Met308. The reduction of nitrate into nitrite occurs in the second step of the mechanism where the two dimethyl-dithiolene ligands have a key role in spreading the excess of negative charge near the Mo atom to make it available for the chemical reaction. The reaction involves the oxidation of the sulfur atoms and not of the molybdenum as previously suggested. The mechanism involves a molybdenum and sulfur-based redox chemistry instead of the currently accepted redox chemistry based only on the Mo ion. The second part of the mechanism involves two protonation steps that are promoted by the presence of MoV species. MoVI intermediates might also be present in this stage depending on the availability of protons and electrons. Once the water molecule is generated only the MoVI species allow water molecule dissociation, and, the concomitant enzymatic turnover.[8]

Regulation

Nitrate Reductase (NR) is regulated at the transcriptional and translational levels induced by light, nitrate, and possibly a negative feedback mechanism. First, nitrate assimilation is initiated by the uptake of nitrate from the root system, reduced to nitrite by nitrate reductase, and then nitrite is reduced to ammonia by nitrite reductase. Ammonia then goes into the GS-GOGAT pathway to be incorporated into amino acids[9]. When the plant is under stress, instead of reducing nitrate via NR to be incorporated into amino acids, the nitrate is reduced to nitric oxide which can have many damaging effects on the plant. Thus, the importance of regulating nitrate reductase activity is to limit the amount of nitric oxide being produced.

Inactivation of Nitrate Reductase

The inactivation of nitrate reductase has many steps and many different signals that aid in the inactivation of the enzyme. Specifically in spinach, the very first step of nitrate reductase inactivation is the phosphorylation of NR on the 543-serine residue. The very last step of nitrate reductase inactivation is the binding of the 14-3-3 adapter protein, which is initiated by the presence of Mg2+ and Ca2+[10]

Anoxic conditions

Studies were done measuring the nitrate uptake and nitrate reductase activity in anoxic conditions to see if there was a difference in activity level and tolerance to anoxia. These studies found that nitrate reductase, in anoxic conditions improves the plants tolerance to being less aerated[10]. This increased activity of nitrate reductase was also related to a increase in nitrite release in the roots. The results of this study showed that the dramatic increase in nitrate reductase in anoxic conditions can be directly attributed to the anoxic conditions inducing the dissociation of 14-3-3 protein from NR and the dephosphorylation of the nitrate reductase[10]

Applications

Nitrate reductase activity can be used as a biochemical tool for predicting grain yield and grain protein production.[11][12]

Nitrate reductase promotes amino acid production in tea leaves.[13] Under south Indian conditions, it is reported that tea plants sprayed with various micronutrients (like Zn, Mn and B) along with Mo enhanced the amino acid content of tea shoots and also the crop yield.[14]

References

  1. ^ PDB: 1Q16​; Bertero MG, Rothery RA, Palak M, Hou C, Lim D, Blasco F, Weiner JH, Strynadka NC (September 2003). "Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A". Nature Structural Biology. 10 (9): 681–7. doi:10.1038/nsb969. PMID 12910261.
  2. ^ Marschner, Petra, ed. (2012). Marschner's mineral nutrition of higher plants (3rd ed.). Amsterdam: Elsevier/Academic Press. p. 135. ISBN 9780123849052.
  3. ^ Tavares P, Pereira AS, Moura JJ, Moura I (December 2006). "Metalloenzymes of the denitrification pathway". Journal of Inorganic Biochemistry. 100 (12): 2087–100. doi:10.1016/j.jinorgbio.2006.09.003. PMID 17070915.
  4. ^ Blasco F, Iobbi C, Ratouchniak J, Bonnefoy V, Chippaux M (June 1990). "Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon". Molecular & General Genetics. 222 (1): 104–11. doi:10.1007/BF00283030. PMID 2233673.
  5. ^ Pantel I, Lindgren PE, Neubauer H, Götz F (July 1998). "Identification and characterization of the Staphylococcus carnosus nitrate reductase operon". Molecular & General Genetics. 259 (1): 105–14. doi:10.1007/s004380050794. PMID 9738886.
  6. ^ Tischner R (October 2000). "Nitrate uptake and reduction in higher and lower plants". Plant, Cell and Environment. 23 (10): 1005–1024. doi:10.1046/j.1365-3040.2000.00595.x.
  7. ^ Jones GJ, Morel FM (May 1988). "Plasmalemma redox activity in the diatom thalassiosira: a possible role for nitrate reductase". Plant Physiology. 87 (1): 143–7. doi:10.1104/pp.87.1.143. PMC 1054714. PMID 16666090.
  8. ^ Cerqueira NM, Gonzalez PJ, Brondino CD, Romão MJ, Romão CC, Moura I, Moura JJ (November 2009). "The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase". Journal of Computational Chemistry. 30 (15): 2466–84. doi:10.1002/jcc.21280. PMID 19360810.
  9. ^ Taiz L, Zeiger E, Moller IM, Murphy A (2014). Plant Physiology and Development (6 ed.). Massachusetts: Sinauer Associates, Inc. p. 356. ISBN 978-1-60535-353-1.
  10. ^ a b c Allègre A, Silvestre J, Morard P, Kallerhoff J, Pinelli E (December 2004). "Nitrate reductase regulation in tomato roots by exogenous nitrate: a possible role in tolerance to long-term root anoxia". Journal of Experimental Botany. 55 (408): 2625–34. doi:10.1093/jxb/erh258. PMID 15475378.
  11. ^ Croy LI, Hageman RH (1970). "Relationship of nitrate reductase activity to grain protein production in wheat". Crop Science. 10 (3): 280–285. doi:10.2135/cropsci1970.0011183X001000030021x.
  12. ^ Dalling MJ, Loyn RH (1977). "Level of activity of nitrate reductase at the seedling stage as a predictor of grain nitrogen yield in wheat (Triticum aestivum L.)". Australian Journal of Agricultural Research. 28 (1): 1–4. doi:10.1071/AR9770001.
  13. ^ Ruan J, Wu X, Ye Y, Härdter R (1988). "Effect of potassium, magnesium and sulphur applied in different forms of fertilisers on free amino acid content in leaves of tea (Camellia sinensis L". J. Sci. Food Agric. 76 (3): 389–396. doi:10.1002/(SICI)1097-0010(199803)76:3<389::AID-JSFA963>3.0.CO;2-X.
  14. ^ Venkatesan S (November 2005). "Impact of genotype and micronutrient applications on nitrate reductase activity of tea leaves". J. Sci. Food Agric. 85 (3): 513–516. doi:10.1002/jsfa.1986.

External links

Assimilatory nitrate reductase

Assimilatory nitrate reductase may refer to:

Nitrate reductase (NADH)

Nitrate reductase (NAD(P)H)

Nitrate reductase (NADPH)

Nitrite reductase

FNR regulon

The fnr (fumarate and nitrate reductase) gene of Escherichia coli encodes a transcriptional activator (FNR) which is required for the expression of a number of genes involved in anaerobic respiratory pathways. The FNR (Fumarate and Nitrate reductase Regulatory) protein of E. coli is an oxygen – responsive transcriptional regulator required for the switch from aerobic to anaerobic metabolism.

The fnr gene is expressed under both aerobic and anaerobic conditions and is subject to autoregulation and repression by glucose, particularly during anaerobic growth.

The functional state of FNR is determined by a (rapid) inactivation of FNR by O2, and a slow (constant) reactivation with glutathione as the reducing agent.

Ferredoxin—nitrate reductase

In enzymology, a ferredoxin—nitrate reductase (EC 1.7.7.2) is an enzyme that catalyzes the chemical reaction

nitrite + H2O + 2 oxidized ferredoxin nitrate + 2 reduced ferredoxin + 2 H+

The 3 substrates of this enzyme are nitrite, H2O, and oxidized ferredoxin, whereas its 3 products are nitrate, reduced ferredoxin, and H+.

This enzyme belongs to the family of oxidoreductases, specifically those acting on other nitrogenous compounds as donors with an iron-sulfur protein as acceptor. The systematic name of this enzyme class is nitrite:ferredoxin oxidoreductase. Other names in common use include assimilatory nitrate reductase, nitrate (ferredoxin) reductase, and assimilatory ferredoxin-nitrate reductase. This enzyme participates in nitrogen metabolism. It has 4 cofactors: iron, Sulfur, Molybdenum, and Iron-sulfur.

FnrS RNA

FnrS RNA is a family of Hfq-binding small RNA whose expression is upregulated in response to anaerobic conditions. It is named FnrS because its expression is strongly dependent on fumarate and nitrate reductase regulator (FNR), a direct oxygen availability sensor.A conserved intergenic region between genes ydaN and dbpA was predicted to encode an sRNA, adjacent to where another non-coding RNA (C0343) has been identified. However, northern blot analysis of this 477bp sequence yielded no results. A subsequent tiling array analysis sequencing Hfq-binding sRNA found that the Watson strand did indeed encode an sRNA.

Moco-II RNA motif

The Moco-II RNA motif is a conserved RNA structure identified by bioinformatics. However, only 8 examples of the RNA motif are known. The RNAs are potentially in the 5' untranslated regions of genes related to molybdenum cofactor (Moco), specifically a gene that encodes a molybdenum-binding domain and a nitrate reductase, which uses Moco as a cofactor. Thus the RNA might be involved in the regulation of genes based on Moco levels. Reliable predictions of Moco-II RNAs are restricted to deltaproteobacteria, but a Moco-II RNA might be present in a betaproteobacterial species. The Moco RNA motif is another RNA that is associated with Moco, and its complex secondary structure and genetic experiments have led to proposals that it is a riboswitch. However, the simpler structure of the Moco-II RNA motif (see diagram) is less typical of riboswitches. Moco-II RNAs are typically followed by a predicted rho-independent transcription terminator.

Molybdopterin

Molybdopterins are a class of cofactors found in most molybdenum-containing and all tungsten-containing enzymes. Synonyms for molybdopterin are: MPT and pyranopterin-dithiolate. The nomenclature for this biomolecule can be confusing: Molybdopterin per se contains no molybdenum; rather, this is the name of the ligand (a pterin) that will bind the active metal. After molybdopterin is eventually complexed with molybdenum, the complete ligand is usually called molybdenum cofactor.

Molybdopterin consists of a pyranopterin, a complex heterocycle featuring a pyran fused to a pterin ring. In addition, the pyran ring features two thiolates, which serve as ligands in molybdo- and tungstoenzymes. In some cases, the alkyl phosphate group is replaced by an alkyl diphosphate nucleotide. Enzymes that contain the molybdopterin cofactor include xanthine oxidase, DMSO reductase, sulfite oxidase, and nitrate reductase.

The only molybdenum-containing enzymes that do not feature molybdopterins are the nitrogenases (enzymes that fix nitrogen). These contain an iron-sulfur center of a very different type, which also usually contains molybdenum. However, if molybdenum is present, it is directly bonded to other metal atoms.

Mycobacterium pseudoshottsii

Mycobacterium pseudoshottsii, a slowly growing chromogenic species was isolated from Chesapeake Bay striped bass (Morone saxatilis) during an epizootic of mycobacteriosis.Taxonomic name not approved yet.

Growth characteristics, acid-fastness and 16S rRNA gene sequencing results were consistent with those of the genus Mycobacterium. Biochemical reactions, growth characteristics and mycolic acid profiles (HPLC) resembled those of Mycobacterium shottsii, a non-pigmented mycobacterium also isolated during the same epizootic. Sequencing of the 16S rRNA genes, the gene encoding the exported repeated protein (erp) and the gene encoding the 65 kDa heat-shock protein (hsp65) and restriction enzyme analysis of the hsp65 gene demonstrated that this group of isolates is unique.

Insertion sequences associated with Mycobacterium ulcerans, IS2404 and IS2606, were detected by PCR. These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 °C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 µg ml–1), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide. On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, Mycobacterium pseudoshottsii sp. nov. The type strain, L15T, has been deposited in the American Type Culture Collection as ATCC BAA-883T and the National Collection of Type Cultures (UK) as NCTC 13318T.

N,N-Dimethyl-1-naphthylamine

N,N-Dimethyl-1-naphthylamine is an aromatic amine. It is formally derived from 1-naphthylamine by replacing the hydrogen atoms on the amino group with methyl groups. N,N-Dimethyl-1-naphthylamine is used in the nitrate reductase test to form a red precipitate of Prontosil by reacting with a nitrite-sulfanilic acid complex.

Nitrate reductase (NAD(P)H)

Nitrate reductase (NAD(P)H) (EC 1.7.1.2, assimilatory nitrate reductase, assimilatory NAD(P)H-nitrate reductase, NAD(P)H bispecific nitrate reductase, nitrate reductase (reduced nicotinamide adenine dinucleotide (phosphate)), nitrate reductase NAD(P)H, NAD(P)H-nitrate reductase, nitrate reductase [NAD(P)H2], NAD(P)H2:nitrate oxidoreductase) is an enzyme with systematic name nitrite:NAD(P)+ oxidoreductase. This enzyme catalises the following chemical reaction

nitrite + NAD(P)+ + H2O nitrate + NAD(P)H + H+

Nitrate reductase is an iron-sulfur molybdenum flavoprotein.

Nitrate reductase (NADH)

Nitrate reductase (NADH) (EC 1.7.1.1, assimilatory nitrate reductase, NADH-nitrate reductase, NADH-dependent nitrate reductase, assimilatory NADH: nitrate reductase, nitrate reductase (NADH2), NADH2:nitrate oxidoreductase) is an enzyme with systematic name nitrite:NAD+ oxidoreductase. This enzyme catalyzes the following chemical reaction

nitrite + NAD+ + H2O nitrate + NADH + H+

Nitrate reductase is an iron-sulfur molybdenum flavoprotein.

Nitrate reductase (NADPH)

Nitrate reductase (NADPH) (EC 1.7.1.3, assimilatory nitrate reductase, assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase, NADPH-nitrate reductase, assimilatory NADPH-nitrate reductase, triphosphopyridine nucleotide-nitrate reductase, NADPH:nitrate reductase, nitrate reductase (NADPH2), NADPH2:nitrate oxidoreductase) is an enzyme with systematic name nitrite:NADP+ oxidoreductase. This enzyme catalises the following chemical reaction

nitrite + NADP+ + H2O nitrate + NADPH + H+

Nitrate reductase is an iron-sulfur molybdenum flavoprotein.

Nitrate reductase (cytochrome)

Nitrate reductase (cytochrome) (EC 1.9.6.1, respiratory nitrate reductase, benzyl viologen-nitrate reductase) is an enzyme with systematic name ferrocytochrome:nitrate oxidoreductase. This enzyme catalises the following chemical reaction

2 ferrocytochrome + 2 H+ + nitrate 2 ferricytochrome + nitrite
Nitrate reductase (quinone)

Nitrate reductase (quinone) (EC 1.7.5.1, nitrate reductase A, nitrate reductase Z, quinol/nitrate oxidoreductase, quinol-nitrate oxidoreductase, quinol:nitrate oxidoreductase, NarA, NarZ, NarGHI) is an enzyme with systematic name nitrite:quinone oxidoreductase. This enzyme catalyses the following chemical reaction

nitrate + a quinol nitrite + a quinone + H2O

This is a membrane-bound enzyme which supports [anaerobic respiration] on nitrate.

Nitrate reductase test

The nitrate reductase test is a test to differentiate between bacteria based on their ability or inability to reduce nitrate (NO3−) to nitrite (NO2−) using anaerobic respiration.

Oxidoreductase FAD-binding domain

The oxidoreductase FAD-binding domain is an evolutionary conserved protein domain.

To date, the 3D-structures of the flavoprotein domain of Zea mays nitrate reductase and of pig NADH:cytochrome b5 reductase have been solved. The overall fold is similar to that of ferredoxin:NADP+ reductase: the FAD-binding domain (N-terminal) has the topology of an anti-parallel beta-barrel, while the NAD(P)-binding domain (C-terminal) has the topology of a classical pyridine dinucleotide-binding fold (i.e. a central parallel beta-sheet flanked by 2 helices on each side).

Phytoglobin-NO cycle

The phytoglobin-nitric oxide cycle is a metabolic pathway induced in plants under hypoxic conditions which involves nitric oxide (NO) and phytoglobin (Pgb). It provides an alternative type of respiration to mitochondrial electron transport under the conditions of limited oxygen supply. Phytoglobin in hypoxic plants acts as part of a soluble terminal nitric oxide dioxygenase system, yielding nitrate ion from the reaction of oxygenated phytoglobin with NO. Class 1 phytoglobins are induced in plants under hypoxia, bind oxygen very tightly at nanomolar concentrations, and can effectively scavenge NO at oxygen levels far below the saturation of cytochrome c oxidase. In the course of the reaction, phytoglobin is oxidized to metphytoglobin which has to be reduced for continuous operation of the cycle. Nitrate is reduced to nitrite by nitrate reductase, while NO is mainly formed due to anaerobic reduction of nitrite which may take place in mitochondria by complex III and complex IV in the absence of oxygen, in the side reaction of nitrate reductase, or by electron transport proteins on the plasma membrane. The overall reaction sequence of the cycle consumes NADH and can contribute to the maintenance of ATP level in highly hypoxic conditions.

Pseudomonas sRNA P11

Pseudomonas sRNA P11 is a ncRNA that was predicted using bioinformatic tools in the genome of the opportunistic pathogen Pseudomonas aeruginosa and its expression verified by northern blot analysis. P11 is located between a putative threonine protein kinase and putative nitrate reductase and is conserved in several Pseudomonas species. P11 has a predicted Rho independent terminator at the 3' end but the function of P11 is unknown.

Respiratory nitrate reductase

Respiratory nitrate reductase may refer to:

Nitrate reductase (cytochrome)

Nitrate reductase

Staphylococcus condimenti

Staphylococcus condimenti is a Gram-positive, coagulase-negative member of the bacterial genus Staphylococcus consisting of single, paired, and clustered cocci. Strains of this species were originally isolated from fermenting soy sauce mash and are positive for catalase, urease, arginine dihydrolase, nitrate reductase, beta-galactosidase, and phosphatase activity.Unlike some clinical Staphylococcus isolates and some food-derived strains, S. condimenti has shown no noticeable resistance to antibiotics including lincomycin and penicillin.

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Oxidoreductases: nitrogenous donor (EC 1.7)
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