Nitrate reductase [NADH]

 

This entry represents the NADH dependent nitrate reductase (NR) from plants and fungi, including NIA1 and NIA2 from Arabidopsis. They contain an N-terminal oxidoreductase molybdopterin binding domain and a C-terminal FAD-binding domain. The N-terminal domain is responsible for the reduction of nitrate; the C-terminal domain is responsible for the regeneration of the FAD cofactor. Only the N-terminal domain function is shown here; the C-terminal function can be found in MACiE:862.

 

Reference Protein and Structure

Sequence
P49050 UniProt (1.7.1.3) IPR012137 (Sequence Homologues) (PDB Homologues)
Biological species
Ogataea angusta (Fungus) Uniprot
PDB
2bii - crystal structure of nitrate-reducing fragment of assimilatory nitrate reductase from Pichia angusta (1.7 Å) PDBe PDBsum 2bii
Catalytic CATH Domains
3.90.420.10 CATHdb (see all for 2bii)
Cofactors
Moo2-molybdopterin cofactor(2−) (1)
Click To Show Structure

Enzyme Reaction (EC:1.7.1.3)

hydron
CHEBI:15378ChEBI
+
nitrate
CHEBI:17632ChEBI
+
NADPH(4-)
CHEBI:57783ChEBI
→
water
CHEBI:15377ChEBI
+
NADP(3-)
CHEBI:58349ChEBI
+
nitrite
CHEBI:16301ChEBI
Alternative enzyme names: NADPH-nitrate reductase, NADPH:nitrate reductase, Assimilatory NADPH-nitrate reductase, Assimilatory nitrate reductase, Assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase, Nitrate reductase (NADPH(2)), Triphosphopyridine nucleotide-nitrate reductase, NADPH:nitrate oxidoreductase,

Enzyme Mechanism

Introduction

This entry represents the mechanism of the N-terminal domain of the protein. In the first step of the reaction (which starts with the oxidized Mo(VI) centre), the Mo is reduced by NAD to Mo(IV). In the next step, the nitrate displaces equatorial hydroxo/water ligand on the molybdenum ion. Upon oxidation of the Mo centre to Mo(VI), the bond between the nitrate oxygen and nitrogen is broken, and nitrite is released.

Catalytic Residues Roles

UniProt PDB* (2bii)
Asp269, Asp269, Asn270, Asn270 Asp271(211)B, Asp271(211)B, Asn272(212)B, Asn272(212)B Asp271 and Asn272 play a crucial role for displacing the Mo-hydroxo/water ligand, as well as stabilising the reaction intermediates formed during the course of the reaction. electrostatic stabiliser, transition state stabiliser
Cys137 Cys139(79)B Binds the molybdenum ion of the Mo-MPT cofactor. metal ligand
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

redox reaction, proton transfer, bimolecular nucleophilic substitution, intermediate formation, overall reactant used, decoordination from a metal ion, coordination to a metal ion, intermediate collapse, electron transfer

References

  1. Fischer K et al. (2005), Plant Cell, 17, 1167-1179. Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site. DOI:10.1105/tpc.104.029694. PMID:15772287.
  2. Cerqueira NM et al. (2009), J Comput Chem, 30, 2466-2484. The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase. DOI:10.1002/jcc.21280. PMID:19360810.
  3. Lu G et al. (1995), J Mol Biol, 248, 931-948. Structural Studies on Corn Nitrate Reductase: Refined Structure of the CytochromebReductase Fragment at 2.5 Å, its ADP Complex and an Active-site Mutant and Modeling of the CytochromebDomain. DOI:10.1006/jmbi.1995.0273. PMID:7760334.

Step 1. The reaction cycle starts by NADPH reducing the Moco bound molybdenum. One of the molybdenum bound oxygens turns into a hydroxide group.

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Catalytic Residues Roles

Residue Roles
Cys139(79)B metal ligand
Asp271(211)B electrostatic stabiliser
Asn272(212)B electrostatic stabiliser

Chemical Components

redox reaction, proton transfer

Step 2. The nitrate then displaces the OH molecule from the coordination sphere of molybdenum.

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Catalytic Residues Roles

Residue Roles
Cys139(79)B metal ligand
Asp271(211)B electrostatic stabiliser
Asn272(212)B electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic substitution, intermediate formation, overall reactant used, decoordination from a metal ion, coordination to a metal ion

Step 3. The Mo metal then creates a double bond with the bound nitrate oxygen, releasing nitrite into the solution. This oxidises the Mo back to its (VI) state. The transition state is stabilised by Asp271 and Asn272.

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Catalytic Residues Roles

Residue Roles
Asp271(211)B electrostatic stabiliser, transition state stabiliser
Asn272(212)B electrostatic stabiliser, transition state stabiliser
Cys139(79)B metal ligand

Chemical Components

overall reactant used, intermediate collapse, electron transfer

Step 4. The nitrite leaves the coordination sphere and the cofactor is regenerated completing the cycle.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys139(79)B metal ligand

Chemical Components

coordination to a metal ion
Download: Image, Marvin File

Step 1. The reaction cycle starts by NADPH reducing the Moco bound molybdenum. One of the molybdenum bound oxygens turns into a hydroxide group.

Step 2. The nitrate then displaces the OH molecule from the coordination sphere of molybdenum.

Step 3. The Mo metal then creates a double bond with the bound nitrate oxygen, releasing nitrite into the solution. This oxidises the Mo back to its (VI) state. The transition state is stabilised by Asp271 and Asn272.

Step 4. The nitrite leaves the coordination sphere and the cofactor is regenerated completing the cycle.

Products.

Contributors

Craig Porter, Gemma L. Holliday, Antonio Ribeiro, Marko Babić