Created by Courtney Thomas
Nitrate reductase is a part of a group of molybdenum-containing enzymes responsible for catalyzing two-electron transfer reactions in global carbon, nitrogen, and sulfur cycles (4, 7). Nitrate reductase catalyzes the first and rate-limiting step in nitrate assimilation, which involves reducing nitrate to nitrite. The nitrate reduction pathway of nitrate reductase allows inorganic nitrogen to be incorporated into organic compounds, therefore increasing the amount of reduced nitrogen on Earth (3, 7). Furthermore, in higher plants, fungi, and algae, nitrate reduction serves as a component of the nitrogen-acquisition mechanism (5).
Plant systems contain molecular mechanisms for regulating nitrate reductase activity. The total nitrate-reducing capacity of a plant system depends on the availability of the substrates in the cytoplasm (NAD(P)H and nitrate), the amount of functional nitrate reductase (controlled at the transcriptional level), and the activity level of the functional nitrate reductase. Direct and indirect regulation of nitrate reduction is overall controlled by the overall plant metabolic level by metabolic sensors and signal transduction pathways. Key metabolites include the free glutamine level (and its ratio to free glutamate) and the nitrate level. If the free glutamine level is low and the nitrate level is high, the nitrate reductase level increases, which in turn increases the nitrate-reducing capacity of the plant. If the free glutamine level is high, nitrate reductase activity and nitrate reduction are decreased. In transgenic plants where nitrate reductase mRNA is expressed constitutively and posttranslational control is lost, glutamine levels do not exercise control over nitrate reduction; only the availability of NADH does. Nitrate induces functional nitrate reductase in plants and a plant’s response to nitrate is dependent upon environmental and genetic factors (5). Applications of nitrate reductase have the capacity to improve crop yields by increasing crop nitrate-use efficiency. As a result, the need for the addition of nitrogen fertilizer to crops would also decrease, which in turn reduces nitrate pollution (3, 5).
Nitrate reductase can help combat the worldwide problem of nitrate pollution. Nitrate accumulation in surface and groundwater poses a threat to both the environment and human health (5). Nitrate reductase is used in nitrate detection kits, providing a more environmentally sound nitrate testing method than heavy metal-based assays (3, 9). The development of a nitrate reductase-based nitrate biosensor is also underway. A nitrate biosensor would monitor nitrate levels in water on a real-time basis (5, 9). Cyclic voltammetry experiments have shown that the nitrate reduction fragment of nitrate reductase possesses high specificity and can be driven by an electrical current without a dye mediator, which makes nitrate reductase ideal for nitrate biosensor development (6). Nitrate reductase, in combination with enzymatic processes, is capable of nitrate removal and the purification of nitrate-polluted drinking water (5, 9). In an enzyme denitrification reactor, where electric current through electron-carrying dyes supplied reducing power to the enzymes, the coupling of nitrate reductase with bacterial denitrification enzymes was able to completely remove nitrate (as dinitrogen) without additions to the water. Applications of this technique may be able to be used for potable water treatment without creating a waste-disposal problem (5).
The nitrate reductase monomer is composed of eight distinct sequence regions: (i) N-terminal sequence region, (ii) molybdenum-molybdopterin domain, (iii) dimer interface domain, (iv) Hinge 1, (v) cytochrome (Cyt) b domain, (vi) Hinge 2, (vii) flavin adenine dinucleotide (FAD) binding domain, and (vii) nicotinamide adenine dinucleotide (NAD(P)H) binding domain (6, 9). The domains are structurally and functionally independent and are linearly arrayed (6). The fragment of interest contains only molybdenum-molybdopterin domain (molybdenum cofactor domain) and the dimer interface (dimerization) domain. The first 25 residues of the N-terminal region were degraded and the sequence terminates in the middle of the Hinge 1 domain (6, 7).
Chain A of a eukaryotic assimilatory nitrate reductase from Pichia angusta (PDB ID = 2BIH), the fragment of interest, was crystallized by x-ray diffraction and has been referred to as the molybdenum-containing nitrate reducing fragment of nitrate reductase or simplified nitrate reductase (6, 7). Simplified nitrate reductase contains the ligand (molybdopterin-S,S)-dioxo-thio-molybdenum(IV) and has the specific function of reducing nitrate to nitrite. The simplified nitrate reductase monomer has a molecular weight of 54,317.97 Daltons and an isoelectric point (pI) of 6.05. Simplified nitrate reductase is only active as a dimer (7).
One asymmetric unit of simplified nitrate reductase contains one monomer with residues 26 to 478, one molybdenum cofactor, and fifty-nine water molecules (7). The secondary structure is 18% helical (18 helices, 86 residues), 28% beta sheet (32 strands, 137 residues), and 54% random coils (2). The monomer has a slightly elongated shape with a mixed alpha and beta structure. The two domains of the monomer, the N-terminal molybdenum cofactor domain and the C-terminal dimerization domain, are clearly defined (7).
The molybdenum cofactor domain, residues 38 to 274, has a secondary structure of thirteen beta strands, seven alpha helices, and four 3/10 helices. The beta strands are organized in one mixed and two antiparallel beta sheets. A three-stranded antiparallel beta sheet located at the N-terminal leads into the mixed beta sheet, which is composed of five beta strands and is located at the opposite end of the domain. A five-stranded antiparallel beta sheet is the central motif, and nine short helices separate it from the mixed beta sheet. A long hairpin loop with a short alpha helix inserted between two beta strands provides the hydrophobic and polar interactions with the C-terminal dimerization domain (7).
The C-terminal dimerization domain core, residues 275 to 448, is formed by two large antiparallel beta sheets, one composed of four beta strands and the other composed of six beta strands. An extension located near the molybdenum cofactor central motif is composed an antiparallel beta sheet and two helices. The N-terminal region of the dimerization domain is an extended loop composed of three helices. The C-terminus that leads into the next sequence region contains one helix (7).
The native state of simplified nitrate reductase is a homodimer (two asymmetric subunits), which is the active state, and has a tendency to dimerize to a homotetramer (7, 9). In the crystal form, tightly associated dimers were formed along twofold crystallographic symmetry axes (7). The dimerization domain is an independently folded region and is significant, because it forms a stable and active dimer (6, 9). The dimer interface extends over 12% of the accessible surface area of each subunit. The dimer is stabilized by 39% polar and 61% nonpolar residues, with 1.6 hydrogen bonds per 75 Å2 of polar dimer interface. Overall, thirty-six hydrogen bonds and salt bridges are responsible for direct intersubunit contacts. The C-terminal domain is the main mediator of dimerization, but interactions between the molybdenum cofactor domains are present (7). Together, the molybdenum cofactor domain and the dimerization domain comprise the nitrate-reducing active site of simplified nitrate reductase (9).
The ligand of simplified nitrate reductase, (molybdopterin-S,S)-dioxo-thio-molybdenum(IV) (see Images tab), is known as the molybdenum cofactor and consists of a mononuclear molybdenum atom (a metal ion) coordinated to the sulfur atoms of molybdopterin, a pterin derivative (4, 5). The molybdenum cofactor is deeply buried within the molybdenum cofactor domain and forms fourteen hydrogen bonds to the main chain and to the side chain atoms of the molybdenum cofactor domain residues. The following residues are involved in the coordination of the molybdenum cofactor: Phe-87, Arg-89, His-91, Cys-139, Ala-140, Asp-195, Arg-238, Gly-247, Ser-249, and Lys-251. Molybdenum is coordinated in a square pyramidal geometry by two sulfur atoms from the molybdopterin (equatorial plane), one sulfur from Cys-139 (equatorial plane), one equatorial oxygen atom, and one axial oxygen stabilized that is stabilized by hydrogen bonds to Ala-140 and Gly-247 (7). Molybdenum must be bound for the molybdenum cofactor to function (5). The dioxo molybdenum center can either be assigned a reduced state (Mo(IV)) or an oxidized state (Mo(VI)) (7). Cycling between these two states is important to the nitrate reduction reaction (4).
The following reaction is catalyzed by eukaryotic assimilatory nitrate reductase:
NO3- + NADH → NO2- + NAD+ + OH-
This reaction has a free energy of -143 kJ/mol; such a large negative value indicates it is practically an irreversible reaction (5). The molybdenum cofactor of simplified nitrate reductase accomplishes nitrate (substrate) reduction to nitrite. Nitrate binds to the reduced molybdenum (Mo(IV)), causing the oxygen closest to molybdenum to attack the metal center. This action displaces the equatorial hydroxo/water ligand (the aforementioned equatorial oxygen ligand), and the reaction intermediate is formed. The residues Asp-271 and Asn-272 coordinate the bound nitrate, and an additional contact to the reaction intermediate might form with Met-427. Asp-271, Asn-272 and Met-427 are functionally important during catalysis, because they are involved in the displacement of the hydroxo/water ligand and the coordination and stabilization of the reaction intermediate. A second molybdenum-oxygen bond is then formed when electrons of the molybdenum d-orbital flip over to the molybdenum-oxygen(nitrate) bond and molybdenum becomes oxidized (Mo(VI)). The oxidation of molybdenum is accompanied by the release of nitrite coordinated by Arg-144. Molybdenum is regenerated to its reduced state by two electrons, derived from a FAD cofactor, transported via an intramolecular electron transport chain (Cyt b domain) (7). Various experiments have suggested that the internal electron transfer is the rate-limiting step in nitrate reductase catalysis. It appears that the rate of nitrate reduction by the molybdenum cofactor is two to three times faster than the rate of the internal electron transfer (9).
Like nitrate reductase, sulfite oxidase is a molybdenum cofactor-containing enzyme (4). Sulfite oxidase catalyzes the oxidation of sulfite to sulfate and performs the following reaction:
SO32- + H2O ↔ SO42- + 2H+
Sulfite oxidase and nitrate reductase are in the same family of molybdenum cofactor-containing enzymes, because they catalyze the transfer of an oxygen atom to or from an electron lone pair of a sulfur or nitrogen atom of the substrate (4). The substrate funnel of both enzymes possesses positively charged residues, because the enzymes act upon anions. However, nitrate reductase also contains a significant patch of negative charge right above the entrance to the active site. The substrate binding cavity of nitrate reductase also narrows into an elongated slot. This space is large enough for the planar nitrate molecule, but not for the bulky sulfate molecule. Therefore sulfate does not inhibit nitrate reductase, but nitrate can inhibit sulfite oxidase (7).
In a structural comparison with plant sulfite oxidase and chicken sulfite oxidase, simplified nitrate reductase displays both conservation and uniqueness of residues (7). Plant sulfite oxidase from Arabidopsis thaliana (PDB ID = 1OGP) has the following associated ligands: cesium ion, glycerol, and (molybdopterin-S,S)-dioxo-thio-molybdenum(IV) (10). The PSI-BLAST results reported an E value of 6e-59 and 32% sequence identity for chain A of plant sulfite oxidase, showing a similarity in primary structure. The DALI results reported a Z score of 43.8 for chain A of plant sulfite oxidase, showing significant similarity in secondary and tertiary structure (1). Chicken (Gallus gallus) sulfite oxidase (PDB ID = 2A9D) has the following associated ligands: molybdenum atom, sulfate ion, and phosphonic acidmono-(2-amino-5,6-dimercapto-4-oxo-3,7,8A,9,10,10A-hexahydro-4H-8-oxa-1,3,9,10-tetraaza-anthracen-7-ylmethyl)ester (8). The PSI-BLAST results reported an E value of 9e-44 and 32% sequence identity for chain A of chicken sulfite oxidase, showing a similarity in primary structure. The DALI results reported a Z score of 46.2 for chain A of chicken sulfite oxidase, showing significant similarity in secondary and tertiary structure (1). Although the sequence identity percentages for both plant sulfite oxidase and chicken sulfite oxidase are less than 50%, the molybdenum cofactor-containing fragments of sulfite oxidase and nitrate reductase are almost 50% identical in sequence (5).
The three cis-peptides His-63-Pro-64, Lys-235-Pro-236, and Lys-309-Pro-310 in simplified nitrate reductase were found to be homologous to Glu-26-Pro-27 and Phe-204-Pro-205 in plant sulfite oxidase and Lys-113-Pro-114, Phe-285-Pro286, and Gln-353-Pro-354 in chicken sulfite oxidase. The Arg-144, Trp-158, and Arg-89 residues of simplified nitrate reductase showed conservation when superimposed with the plant sulfite oxidase and chicken sulfite oxidase counterparts. A unique characteristic of the sulfite oxidase family of enzymes, simplified nitrate reductase, plant sulfite oxidase, and chicken sulfite oxidase all have Cys-139 serving as a sulfur ligand of molybdenum. Asn-272 and Met-427 are two nitrate reductase-conserved residues, and the Thr-425 residue seems to be unique to simplified nitrate reductase and is probably is involved in nitrate binding and catalysis. The Asn-272 residue of simplified nitrate reductase is replaced by a Tyr-322 residue in chicken sulfite oxidase, which is conserved among sulfite oxidases and has shown to be involved in intramolecular-coupled electron/proton transfer and substrate binding and catalysis. The Arg-374 residue in plant sulfite oxidase and the Arg-450 residue in chicken sulfite oxidase appear to be important for substrate/product binding. In simplified nitrate reductase, the arginine residue is replaced by Thr-425. The simplified nitrate reductase active site Met-427 residue replaces a valine in plant sulfite oxidase and chicken sulfite oxidase. Unlike a comparable sulfite oxidase-conserved arginine residue, methionine does not have the possibility to participate as an electron donor in hydrogen bonds with its side chain (7).
Simplified nitrate reductase can also be compared to a higher resolution molybdenum cofactor-containing fragment of nitrate reductase from Pichia angusta (PDB = 2BII). The monomer of the higher resolution structure contains a molybdenum cofactor, one sodium ion, two sulfate ions, and two glycerol molecules (the first 63 residues were degraded). Sulfate-induced conformational changes in the high resolution nitrate reductase were compared to simplified nitrate reductase. In the high resolution nitrate reductase, one out of two sulfate molecules was in close to the active site, near the narrowing substrate entrance. Changes in conformation affected the active site residues and were close to the sulfate binding site. The Phe-156 residue showed a large rotation in the phenyl group. The Phe-156 residue, along with Asn-66 and Arg-89, directly coordinate sulfate. The active site residues Arg-144, Asp-271, Trp-158, and Ser-426 were slightly distorted toward the bound sulfate. The side chain of the Met-427 residue in the high resolution nitrate reductase pointed toward the molybdenum, whereas in simplified nitrate reductase, the Met-427 side chain turns away from the active site. These conformational changes due to sulfate might be related to the activation of nitrate reductase by phosphate. Further studies are needed to determine the functionality of sulfate and phosphate in relation to nitrate reductase activity (7).