Products

Enzymes for Research, Diagnostic and Industrial Use

Nitrate reductase

Official Full Name
Nitrate reductase
Background
Nitrate reductase (NADH) is an enzyme with system name nitrite:NAD+ oxidoreductase. This enzyme catalises the following chemical reaction:nitrite + NAD+ + H2O↔ nitrate + NADH + H+. Nitrate reductase us an iron-sulfur molybdenum flavoprotein.
Synonyms
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

Catalog
ProductName
EC No.
CAS No.
Source
Price
CatalogEXWM-1688
EC No.EC 1.9.6.1
CAS No.9029-42-9
Source
CatalogEXWM-1609
EC No.EC 1.7.1.2
CAS No.9029-27-0
Source
CatalogEXWM-1602
EC No.EC 1.7.1.1
CAS No.9013-03-0
Source
CatalogNATE-0998
EC No.EC 1.7.1.2
CAS No.9029-27-0
SourceAspergillus sp.
CatalogNATE-0474
EC No.EC 1.7.1.1
CAS No.9013-03-0
SourceCorn
CatalogNATE-0486
EC No.EC 1.7.1.1
CAS No.9013-03-0
SourcePichia pastoris
CatalogNATE-0484
EC No.EC 1.7.1.2
CAS No.9029-27-0
SourceAspergillus nig...
CatalogNATE-0485
EC No.EC 1.9.6.1
CAS No.9029-42-9
SourceEscherichia col...
Related Services
Related Protocols
NITRATE REDUCTASE(CYTOCHROME) -Enzymatic Assay Protocol
Related Reading

Nitrate reductases (NR, EC 1.6.6.1-3), the first enzyme for nitrate assimilation, is a multi-domain protein capable of mediating the donation of electrons from NAD(P)H to artificial acceptors and redox proteins. The enzymatic reduction of nitrate (NO3−) to nitrite (NO2−) by NR is critical for nitrogen acquisition in most crop plants, since its main substrate nitrate is required for signaling and serves as the primary nitrogen source in fertilized soils. NR is a central point in plants, algae, and fungi for integration of metabolism by governing flux of reduced nitrogen through several regulatory mechanisms. NR consists of prosthetic groups molybdopterin, Fe-heme, and FAD (flavin adenine dinucleotide) in a 1:1:1 stoichiometry, where FAD and Mo-pterin domains are the binding sites for NAD(P)H and NO3−, respectively. The cytochrome b5-like heme domain expedites the electron transfer from FAD domain to active site Mo-pterin.

Structure

Nitrate reductase

The NR monomer is composed of a ~100-kD polypeptide, heme-iron, flavin adenine dinucleotide (FAD), and molybdenum-molybdopterin (Mo-MPT). Eight sequence segments of NR are N-terminal "acidic" region, Mo-MPT domain with nitrate-reducing active site, interface domain, hinge 1 containing serine phosphorylated in reversible activity regulation with inhibition by 14-3-3 binding protein, cytochrome b domain, hinge 2, FAD domain, and NAD(P)H domain, respectively. A three-dimensional dimeric NR structure model has been constructed from structures of sulfite oxidase and cytochrome b reductase.

Nitrate reductaseFigure 1. Structure of nitrate reductase. (Campbell W H. 1996)

It is noteworthy that a transmembrane respiratory NR contains three subunits; an alpha, a beta and two gamma. It serves as the second nitrate reductase to substitute for the anaerobic NR in Escherichia coli,allowing it to function as a proton pump and use nitrate as an electron acceptor during anaerobic respiration. NR gamma subunit is similar to cytochrome b and transfers electrons from quinones to the beta subunit. The NR of higher plants belongs to a cytosolic protein, which has a GPI-anchored variant that is found on the outer face of the plasma membrane.

Classification

Eukaryotic NRs are part of the sulfite oxidase family of molybdoenzymes and they are able to transfer electrons from NADH or NADPH to nitrate.

Prokaryotic NRs are a member of the DMSO reductase family of molybdoenzymes. They have been divided into three groups including assimilatory nitrate reductases (Nas), respiratory nitrate reductase (Nar), and periplasmic nitrate reductases (Nap), whose active site is a Mo ion that is responsible for the four thiolate functions of two pterin molecules. The Mo is coordinated by one amino-acid side chain and oxygen and/or sulfur ligands and is covalently attached to the protein by a cysteine ligand in Nap, and an aspartate in Nar. The exact environment of the Mo ion in certain of these enzymes is still controversial.

Mechanism

Nitrate molecule binds to the active site with the Mo ion at the +6 oxidation state. Electron transfer to the active site is completed only in the proton-electron transfer stage, where the MoV species are critical in catalysis. The present sulfur atom in the molybdenum coordination sphere could generate a pseudo-dithiolene ligand that protects it from being attacked by the solvent. Upon the nitrate binding, a conformational rearrangement of this ring occurs and is stabilized by the conserved methionines Met141 and Met308. This rearrangement enables the direct interaction of the nitrate with MoVI ion. In the second step of the mechanism, the reduction of nitrate into nitrite occurs, involving the oxidation of the sulfur atoms and not of the molybdenum. The two dimethyl-dithiolene ligands are crucial in dispersing the excessive negative charge near the Mo atom to make it available for the chemical reaction. The mechanism implicates a molybdenum and sulfur-based redox chemistry rather than the widely accepted redox chemistry based only on the Mo ion. The second part of the mechanism includes two protonation steps that are facilitated by the presence of MoV species. Depending on the availability of protons and electrons, MoVI intermediates might also be present in this stage. Once the water molecule is produced, only the MoVI species could conduct water molecule dissociation along with the concomitant enzymatic turnover.

Nitrate reductaseFigure 2. . Proposed mechanism for nitrate reduction by respiratory nitrate reductases. (Coelho C; et al. 2015)

Applications

The reduction of nitrate to nitrite by nitrate reductase, and the reduction of nitrite to ammonium by nitrite reductase are two major procedures in the biological conversion of inorganic nitrate to ammonium, an eight-electron reduction process. NR has been proposed as an important enzymatic source of nitric oxide (NO). NR also plays a role in NO homeostasis by transferring electrons from NAD(P)H through its diaphorase/dehydrogenase domain to a truncated hemoglobin THB1 that scavenges NO by its dioxygenase activity, and to the molybdoenzyme NO-forming nitrite reductase (NOFNiR) that is responsible for NO synthesis from nitrite. Homeostasis of the crucial signaling molecule NO in photosynthetic organisms is dependent on two key molybdoenzymes, NR and NOFNiR, as well as on the dioxygenase potency of hemoglobins.

NR activity can be employed as a biochemical tool to predict rain yield and grain protein production. NR also enhances amino acid production in tea leaves. Under south Indian conditions, it has been reported that tea plants sprayed with various micronutrients along with Mo has an increased amino acid content of tea shoots and also an augmented crop yield.

References

  1. Campbell W H. Nitrate reductase biochemistry comes of age. Plant Physiol, 1996, 111:355–361.
  2. Coelho C; et al. Structural and mechanistic insights on nitrate reductases. Protein Sci, 2015, 24:1901—1911.

Sitemap  Privacy Policy  Terms and Conditions
Copyright ©2011 - 2018 Creative Enzymes.
Contact Us 45-1 Ramsey Road, Shirley, NY 11967, USA
Email: info@creative-enzymes.com
Tel: 1-631-562-8517 1-516-512-3133
Fax: 1-631-938-8127
Distributors To view the contact information for a specific location, select the desired country: