Nitrate reductases are molybdoenzymes that reducenitrate (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.
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.
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.
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.
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. 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.
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+
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. 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
Nitrate reductase activity can be used as a biochemical tool for predicting grain yield and grain protein production.
Nitrate reductase promotes amino acid production in tea leaves. 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.
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