View source for Nitrate reductase

{{Short description|Class of enzymes}}
{{other uses}}
{{infobox enzyme
| Name       = nitrate reductase
| EC_number  = 1.7.99.4
| CAS_number = 9013-03-0
| GO_code    = 0009325
| image      = Nitrate reductase.png
| width      =
| caption    = structure of nitrate reductase A from ''[[Escherichia coli|E. coli]]''<ref name="pmid12910261">{{PDB|1Q16}}; {{cite journal | vauthors = Bertero MG, Rothery RA, Palak M, Hou C, Lim D, Blasco F, Weiner JH, Strynadka NC | title = Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A | journal = Nature Structural Biology | volume = 10 | issue = 9 | pages = 681–7 | date = September 2003 | pmid = 12910261 | doi = 10.1038/nsb969 | s2cid = 33272416 }}</ref> 
}}
{{Pfam_box
| Symbol = Molybdopterin
| Name = Molybdopterin oxidoreductase (nitrate reductase alpha subunit)
| image =
| width =
| caption =
| Pfam= PF00384
| InterPro= IPR006656
| SMART=
| Prosite = PDOC00392
| SCOP = 1cxs
| TCDB =
| OPM family= 3
| OPM protein= 1kqf
}}
{{Pfam_box
| Symbol = Fer4_11
| Name = 4Fe-4S dicluster domain<br />(nitrate reductase beta subunit)
| image =
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| Pfam= PF13247
| InterPro=
| SMART=
| Prosite =
| SCOP =
| TCDB =
| OPM family=
| OPM protein=
}}
{{Infobox protein family
| Symbol = Nitrate_red_gam
| Name = Nitrate reductase gamma subunit
| image =
| width =
| caption =
| Pfam = PF02665
| Pfam_clan = 
| InterPro = IPR003816
| SMART =
| PROSITE =
| MEROPS =
| SCOP = 1q16
| TCDB = 5.A.3
| OPM family = 3
| OPM protein = 1q16
| CAZy =
| CDD =
}}
{{Infobox protein family
| Symbol = Nitrate_red_del
| Name = Nitrate reductase delta subunit
| image =
| width =
| caption =
| Pfam = PF02613
| Pfam_clan = 
| InterPro = IPR003765
| SMART =
| PROSITE =
| MEROPS =
| SCOP =
| TCDB =
| OPM family =
| OPM protein =
| CAZy =
| CDD =
}}
{{Pfam_box
| Symbol = NapB
| Name = Nitrate reductase cytochrome c-type subunit (NapB)
| image =
| width =
| caption =
| Pfam= PF03892
| InterPro= IPR005591
| SMART=
| Prosite =         
| SCOP = 1jni
| TCDB =
| OPM family=
| OPM protein=
}}
{{Pfam_box
| Symbol = NapE
| Name = Periplasmic nitrate reductase protein NapE
| image =
| width =
| caption =
| Pfam= PF06796
| InterPro= IPR010649
| SMART=
| Prosite =         
| SCOP =    
| TCDB =
| OPM family=
| OPM protein=
}}
'''Nitrate reductases''' are [[molybdoenzyme]]s that [[redox|reduce]] [[nitrate]] ({{Chem|N|O|3|-}}) to [[nitrite]] ({{Chem|N|O|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.<ref>{{cite book|editor1-last=Marschner|editor1-first=Petra|title=Marschner's mineral nutrition of higher plants|date=2012|publisher=Elsevier/Academic Press|location=Amsterdam|isbn=9780123849052|page=135|edition=3rd}}</ref>

== 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).<ref>{{Cite book|title=Prokaryotic Nitrate Reduction: Molecular Properties and Functional Distinction among Bacterial Nitrate Reductases|last=Moreno-Vivián, Conrado Cabello, Purificación Martínez-Luque, Manuel Blasco, Rafael Castillo, Francisco|publisher=American Society for Microbiology|oclc=678511191}}</ref> The active site of these enzymes is a [[molybdenum]] ion that is bound to the four thiolate functional groups of two [[pterin]] molecules. The coordination sphere of the molybdenum ion is completed by one amino-acid side chain and oxygen and/or sulfur [[ligand]]s. In Nap, the molybdenum is covalently attached to the protein by a cysteine side chain, and an aspartate side chain in Nar.<ref name="pmid17070915">{{cite journal | vauthors = Tavares P, Pereira AS, Moura JJ, Moura I | title = Metalloenzymes of the denitrification pathway | journal = Journal of Inorganic Biochemistry | volume = 100 | issue = 12 | pages = 2087–100 | date = December 2006 | pmid = 17070915 | doi = 10.1016/j.jinorgbio.2006.09.003 }}</ref>

== Structure ==
Prokaryotic nitrate reductases have two major types, transmembrane nitrate reductases (NAR) and periplasmic nitrate reductases (NAP). NAR allows for proton translocation across the cellular membrane and can contribute to the generation of ATP by the [[Chemiosmosis#Proton-motive force|proton motive force]]. NAP cannot do so.<ref>{{cite journal | vauthors = Kuypers MM, Marchant HK, Kartal B | title = The microbial nitrogen-cycling network | journal = Nature Reviews. Microbiology | volume = 16 | issue = 5 | pages = 263–276 | date = May 2018 | pmid = 29398704 | doi = 10.1038/nrmicro.2018.9 | s2cid = 3948918 | hdl = 21.11116/0000-0003-B828-1 | hdl-access = free }}</ref>

The transmembrane respiratory nitrate reductase<ref>{{cite web|url=http://expasy.org/enzyme/1.7.99.4 |title=ENZYME entry: EC 1.7.99.4 |website=ENZYME Enzyme nomenclature database |access-date=25 April 2019}}</ref> is composed of three subunits; an 1 alpha, 1 beta and 2 gamma. It can substitute for the NRA enzyme in ''[[Escherichia coli]]'', allowing it to use nitrate as an [[electron acceptor]] for anaerobic respiration.<ref name="pmid2233673">{{cite journal | vauthors = Blasco F, Iobbi C, Ratouchniak J, Bonnefoy V, Chippaux M | title = Nitrate reductases of Escherichia coli: sequence of the second nitrate reductase and comparison with that encoded by the narGHJI operon | journal = Molecular & General Genetics | volume = 222 | issue = 1 | pages = 104–11 | date = June 1990 | pmid = 2233673 | doi = 10.1007/BF00283030 | s2cid = 22797628 }}</ref> A transmembrane nitrate reductase that can function as a proton pump (similar to the case of [[anaerobic respiration]]) has been discovered in the [[diatom]] ''[[Thalassiosira weissflogii]]''.<ref name="Jones_1988">{{cite journal | vauthors = Jones GJ, Morel FM | title = Plasmalemma redox activity in the diatom thalassiosira: a possible role for nitrate reductase | journal = Plant Physiology | volume = 87 | issue = 1 | pages = 143–7 | date = May 1988 | pmid = 16666090 | pmc = 1054714 | doi = 10.1104/pp.87.1.143 }}</ref>

The nitrate reductase of higher plants, algae, and fungi is a homodimeric cytosolic protein with five conserved domains in each monomer: 1) an Mo-MPT domain that contains the single [[molybdopterin]] cofactor, 2) a dimer interface domain, 3) a cytochrome b domain, and 4) an NADH-binding domain that combines with 5) an FAD-binding domain to form the cytochrome b reductase fragment.<ref>{{cite journal | vauthors = Campbell WH | title =  Nitrate Reductase Structure, Function and Regulation: Bridging the Gap between Biochemistry and Physiology | journal = Annual Review of Plant Physiology and Plant Molecular Biology | volume = 50 | issue = 1 | pages = 277–303 | date = June 1999 | pmid = 15012211 | doi = 10.1146/annurev.arplant.50.1.277 | s2cid = 22029078 }}</ref> There exists a [[Glycophosphatidylinositol]]-anchored variant that is found on the outer face of the plasma membrane. Its function is not clear.<ref name=Tischner_2000>{{cite journal| vauthors = Tischner R |title=Nitrate uptake and reduction in higher and lower plants |journal=Plant, Cell and Environment |date=October 2000 |volume=23 |issue=10 |pages=1005–1024 |doi=10.1046/j.1365-3040.2000.00595.x|doi-access=free |bibcode=2000PCEnv..23.1005T }}</ref>{{Update inline|date=February 2024|?=yes|reason=23 y/o reference}}

== Mechanism ==
In prokaryotic periplasmic nitrate reductase, the nitrate anion binds to Mo(IV).  Oxygen transfer yields an Mo(VI) oxo intermediate with release of nitrite.  Reduction of the Mo oxide and protonolysis removes the oxo group, regenerating Mo(IV).<ref>{{cite journal |  doi=10.1021/cr400443z| title=The Mononuclear Molybdenum Enzymes| year=2014| last1=Hille| first1=Russ| last2=Hall| first2=James| last3=Basu| first3=Partha| journal=Chemical Reviews| volume=114| issue=7| pages=3963–4038| pmid=24467397| pmc=4080432}}</ref>

Similar to the prokaryotic nitrate reduction mechanism, in eukaryotic nitrate reductase, an oxygen in nitrate binds to Mo in the +4 oxidation state, displacing a hydroxide ion. Then the Mo d-orbital electrons flip over, creating a multiple bond between Mo(VI) and that oxygen, ejecting nitrite. The Mo(VI) double bond to oxygen is reduced by NAD(P)H passed through the intramolecular transport chain.<ref>{{cite journal | vauthors = Fischer K, Barbier GG, Hecht HJ, Mendel RR, Campbell WH, Schwarz G | title = Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site | journal = The Plant Cell | volume = 17 | issue = 4 | pages = 1167–79 | date = April 2005 | pmid = 15772287 | pmc = 1087994 | doi = 10.1105/tpc.104.029694 | bibcode = 2005PlanC..17.1167F }}</ref>

== 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.<ref name="Taiz et al.">{{cite book|title=Plant Physiology and Development|last1=Taiz|first1=Lincoln|last2=Zeiger|first2=Eduardo|last3=Moller|first3=Ian Max|last4=Murphy|first4=Angus | name-list-style = vanc |date=2014|publisher=Sinauer Associates, Inc.|isbn=978-1-60535-353-1|edition=6|location=Massachusetts|page=356 }}</ref> 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 Mg<sup>2+</sup> and Ca<sup>2+</sup>.<ref name="Allegre et al.2">{{cite journal | vauthors = Allègre A, Silvestre J, Morard P, Kallerhoff J, Pinelli E | title = Nitrate reductase regulation in tomato roots by exogenous nitrate: a possible role in tolerance to long-term root anoxia | journal = Journal of Experimental Botany | volume = 55 | issue = 408 | pages = 2625–34 | date = December 2004 | pmid = 15475378 | doi = 10.1093/jxb/erh258 | url = https://hal.archives-ouvertes.fr/hal-00987393/file/allegre_4102.pdf | doi-access = free }}</ref> Higher plants and some algae post-translationally regulate NR by phosphorylation of serine residues and subsequent binding of a 14-3-3 protein.<ref>{{cite journal | vauthors = Wang Y, Bouchard JN, Coyne KJ | title = Expression of novel nitrate reductase genes in the harmful alga, Chattonella subsalsa | journal = Scientific Reports | volume = 8 | issue = 1 | pages = 13417 | date = September 2018 | pmid = 30194416 | pmc = 6128913 | doi = 10.1038/s41598-018-31735-5 | bibcode = 2018NatSR...813417W }}</ref>

=== 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.<ref name="Allegre et al.2" /> This increased activity of nitrate reductase was also related to an 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.<ref name="Allegre et al.2" />

== Applications ==
Nitrate reductase activity can be used as a biochemical tool for predicting grain yield and grain protein production.<ref name="Croy_Hageman_1970">{{cite journal |vauthors=Croy LI, Hageman RH | title = Relationship of nitrate reductase activity to grain protein production in wheat | journal = Crop Science | volume = 10 | issue = 3| pages = 280–285 | year = 1970 | doi =10.2135/cropsci1970.0011183X001000030021x  }}</ref><ref name="Dalling_Loyn_1977">{{cite journal |vauthors=Dalling MJ, Loyn RH | title = Level of activity of nitrate reductase at the seedling stage as a predictor of grain nitrogen yield in wheat (Triticum aestivum L.) | journal = Australian Journal of Agricultural Research | volume = 28 | issue = 1 | pages = 1–4 | year = 1977 | doi =  10.1071/AR9770001 }}</ref>

Nitrate reductase can be used to test nitrate concentrations in biofluids.<ref>{{Cite journal|last=Mori|first=Hisakazu|date=2001|title=Determination of Nitrate in Biological Fluids Using Nitrate Reductase in a Flow System.|journal=Journal of Health Science|volume=47|issue=1|pages=65–67|doi=10.1248/jhs.47.65|issn=1344-9702|doi-access=free}}</ref>

Nitrate reductase promotes amino acid production in tea leaves.<ref name="Ruan_1988">{{cite journal |vauthors=Ruan J, Wu X, Ye Y, Härdter R | title = Effect of potassium, magnesium and sulphur applied in different forms of fertilisers on free amino acid content in leaves of tea (Camellia sinensis L | journal = J. Sci. Food Agric. | volume = 76 | issue =  3 | pages = 389–396 | year = 1988 | doi =10.1002/(SICI)1097-0010(199803)76:3<389::AID-JSFA963>3.0.CO;2-X   }}</ref> 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.<ref name="Venkatesan_2005">{{cite journal | vauthors = Venkatesan S | title = Impact of genotype and micronutrient applications on nitrate reductase activity of tea leaves | journal = J. Sci. Food Agric. | volume = 85 | issue =  3 | pages = 513–516 |date=November 2005 | doi =  10.1002/jsfa.1986 | bibcode = 2005JSFA...85..513V }}</ref>
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== References ==
{{Reflist|32em}}

== External links ==
* {{UMichOPM|protein|pdbid|1q16}}
* {{MeshName|Nitrate+reductases}}

{{Nitrogenous donor oxidoreductases}}
{{Enzymes}}
{{Nitric oxide signaling}}
{{Portal bar|Biology|border=no}}

{{Flavoproteins}}
{{InterPro content|IPR003816}}

{{DEFAULTSORT:Nitrate Reductase}}
[[Category:Enzymes]]
[[Category:Integral membrane proteins]]
[[Category:EC 1.7.99]]
[[Category:Protein families]]