Oxygen insensitive NAD(P)H nitroreductase
Nitroreductase (NTR) isolated from Escherichia coli catalyses the reduction of nitroaromatics, such as nitrofurazone and nitrofurantoin, to hydroxylamines and quinones, such as menadione, to quinols and has potential uses in chemotherapy and bioremediation. Unusually, it can use either NADPH or NADH as the reducing agent. The enzyme is oxygen-insensitive in that it does not transfers electrons to oxygen to produce superoxide. NTR is part of a family of FMN-containing oxidoreductases that have broadly similar substrate specificities. These enzymes are dimeric and contain two active sites, catalysing nitroaromatic reduction via a ping-pong bi bi mechanism. The substrate binds over the pyrimidine and central rings of the flavin. A portion of helix H6 can flex to accommodate the differently sized inhibitors suggesting a mechanism for accommodating varied substrates. This enzyme was first isolated from bacteria growing in a weapons storage dump, and can reduce trinitrotoluene.
Some nitro containing antibiotics depend on activation by bacterial NTR. The native enzyme shows that the principal structural changes occur in the FMN cofactor and indicate that the enzyme itself is a relatively rigid structure that primarily provides a rigid structural framework on which hydride transfer occurs. NTR is of interest in suicide gene therapy, where NTR is used as an activating enzyme for nitroaromatic prodrugs. The hydroxylamines produced can target proteins and DNA, causing strand cleavage in the latter. They can also be further activated to produce DNA cross-linking.
Reference Protein and Structure
- Sequence
-
P38489
(1.-.-.-, 1.5.1.34)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Escherichia coli K-12 (Bacteria)
- PDB
-
1idt
- STRUCTURAL STUDIES ON A PRODRUG-ACTIVATING SYSTEM-CB1954 AND FMN-DEPENDENT NITROREDUCTASE
(2.0 Å)
- Catalytic CATH Domains
-
3.40.109.10
(see all for 1idt)
- Cofactors
- Fmnh2(2-) (2)
Enzyme Mechanism
Introduction
This enzyme derives reducing equivalents from NADH, NADPH, or other nicotinamides by means of a flavin mononucleotide cofactor (FMN). The reaction proceeds by reduction of the nitro group of the prodrug substrate, forming a hydroxylamine product in a ping-pong Bi-Bi reaction pathway.
Prior to the substrate binding, FMN accepts a hydride from NAD(P)H onto N1 or N5. NAD(P)H then leaves as NAD(P)+. The resultant negative charge is transferred through the delocalised system onto O3 where electrostatic interactions with K14 and K74 stabilise it. The nitro group to be attacked stacks above FMN which then transfers the hydride. At the same time the nitro group accepts a proton from water . The resultant -N(OH)2 group spontaneously eliminates water. A second hydride is passed to FMN by NAD(P)H, the charge again moves to O3 where it is best stabilised followed by transfer of the hydride to the nitroso group which simultaneously accepts a proton from water onto the oxygen, leading to the hydroxylamine product.
Catalytic Residues Roles
| UniProt | PDB* (1idt) | ||
| Lys14 | Lys14A | Lys14 is thought to stabilise the negative charge on N1 and O3 of FMN during its transient reduction. It is also thought to modulate the redox potential of FMN. | hydrogen bond donor, electrostatic stabiliser |
| Lys74 | Lys74A | Lys74 is thought to stabilise the negative charge on O3 of FMN during its transient reduction. It is also thought to modulate the redox potential of FMN. | hydrogen bond donor, electrostatic stabiliser |
| Glu165 (main-N) | Glu165A (main-N) | Forms a hydrogen bond to N5 of FMN and is thought to increase its oxidative power. | hydrogen bond donor, electrostatic stabiliser |
Chemical Components
hydride transfer, aromatic unimolecular elimination by the conjugate base, aromatic bimolecular nucleophilic addition, cofactor used, proton transfer, bimolecular nucleophilic addition, native state of cofactor regenerated, overall reactant used, intermediate formation, intramolecular elimination, dehydration, native state of enzyme regeneratedReferences
- Johansson E et al. (2003), J Med Chem, 46, 4009-4020. Studies on the Nitroreductase Prodrug-Activating System. Crystal Structures of Complexes with the Inhibitor Dicoumarol and Dinitrobenzamide Prodrugs and of the Enzyme Active Form. DOI:10.1021/jm030843b. PMID:12954054.
- Bai J et al. (2015), Chembiochem, 16, 1219-1225. Altering the Regioselectivity of a Nitroreductase in the Synthesis of Arylhydroxylamines by Structure-Based Engineering. DOI:10.1002/cbic.201500070. PMID:25917861.
- Race PR et al. (2005), J Biol Chem, 280, 13256-13264. Structural and Mechanistic Studies of Escherichia coli Nitroreductase with the Antibiotic Nitrofurazone: REVERSED BINDING ORIENTATIONS IN DIFFERENT REDOX STATES OF THE ENZYME. DOI:10.1074/jbc.m409652200. PMID:15684426.
- Haynes CA et al. (2002), J Biol Chem, 277, 11513-11520. Structures of Nitroreductase in Three States: EFFECTS OF INHIBITOR BINDING AND REDUCTION. DOI:10.1074/jbc.m111334200. PMID:11805110.
- Lovering AL et al. (2001), J Mol Biol, 309, 203-213. The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 Å, 1.8 Å and 2.4 Å resolution. DOI:10.1006/jmbi.2001.4653. PMID:11491290.
- Parkinson GN et al. (2000), J Med Chem, 43, 3624-3631. Crystal Structure of FMN-Dependent Nitroreductase fromEscherichiacoliB: A Prodrug-Activating Enzyme†,‡. DOI:10.1021/jm000159m. PMID:11020276.
Step 1. NAD initiates an elimination of a hydride ion, which attacks the FMN in a nucleophilic addition.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Lys14A | hydrogen bond donor, electrostatic stabiliser |
| Lys74A | hydrogen bond donor, electrostatic stabiliser |
| Glu165A (main-N) | electrostatic stabiliser |
Chemical Components
hydride transfer, ingold: aromatic unimolecular elimination by the conjugate base, ingold: aromatic bimolecular nucleophilic addition, cofactor used
Step 2. FMN eliminates the hydride, which attacks the nitro group of the substrate in a nucleophilic addition. The intermediate then deprotonates a water molecule.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Lys14A | hydrogen bond donor, electrostatic stabiliser |
| Lys74A | hydrogen bond donor, electrostatic stabiliser |
| Glu165A (main-N) | electrostatic stabiliser |
Chemical Components
hydride transfer, proton transfer, ingold: bimolecular nucleophilic addition, ingold: aromatic unimolecular elimination by the conjugate base, native state of cofactor regenerated, overall reactant used, intermediate formation
Step 3. The intermediate eliminates water from itself, forming a nitroso intermediate.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Lys14A | hydrogen bond donor |
| Lys74A | hydrogen bond donor |
| Glu165A (main-N) | hydrogen bond donor |
Chemical Components
proton transfer, ingold: intramolecular elimination, dehydration
Step 4. NAD initiates an elimination of a hydride ion, which attacks the FMN in a nucleophilic addition.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Lys14A | hydrogen bond donor, electrostatic stabiliser |
| Lys74A | hydrogen bond donor, electrostatic stabiliser |
| Glu165A (main-N) | electrostatic stabiliser |
Chemical Components
hydride transfer, ingold: aromatic unimolecular elimination by the conjugate base, ingold: aromatic bimolecular nucleophilic addition, cofactor used
Step 5. FMN eliminates the hydride, which attacks the nitroso group of the intermediate in a nucleophilic addition. The intermediate then deprotonates a water molecule to produce the product.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Glu165A (main-N) | electrostatic stabiliser |
| Lys14A | hydrogen bond donor, electrostatic stabiliser |
| Lys74A | hydrogen bond donor, electrostatic stabiliser |
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