Database accession: MF7000512
Name: Prodrug-Activating-System CB1954 with FMN-dependent nitroreductase
PDB ID: 1idt
Experimental method: X-ray (2.00 Å)
Assembly: Homodimer
Source organism: Escherichia coli
Primary publication of the structure:
Johansson E, Parkinson GN, Denny WA, Neidle S
Studies on the nitroreductase prodrug-activating system. Crystal structures of complexes with the inhibitor dicoumarol and dinitrobenzamide prodrugs and of the enzyme active form.
(2003) J. Med. Chem. 46: 4009-20
PMID: 12954054
Abstract:
The E. coli nitroreductase enzyme (NTR) has been widely used in suicide gene therapy (GDEPT and ADEPT) applications as a activating enzyme for nitroaromatic prodrugs of the dinitrobenzamide class. NTR has been previously shown to be a homodimeric enzyme with two active sites. We present here the crystal structures of the reduced form of NTR and its complexes with the inhibitor dicoumarol and three dinitrobenzamide prodrugs. Comparison of the structures of the oxidized and reduced forms of 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. The aziridinyldinitrobenzamide prodrug CB 1954 binds in nonidentical ways in both of the two active sites of the homodimeric enzyme, employing both hydrophobic and (in active site B) a direct H-bond contact to the side chain of Lys14. In active site A the 2-nitro group stacks above the FMN, and in active site B the 4-nitro group does, explaining why reduction of either nitro group is observed. In contrast, the larger mustard group of the dinitrobenzamide mustard compound SN 23862 forces the prodrug to bind at both active sites with only the 2-nitro group able to participate in hydride transfer from the FMN, explaining why only the 2-hydroxylamine reduction product is observed. In each site, the nitro groups of the prodrug make direct H-bond contacts with the enzyme; in active Site A the 2-nitro to Ser40 and the 4-nitro to Asn71, while in active Site B the 2-nitro contacts the main chain nitrogen of Thr41 and the 4-nitro group the Lys14 side chain. The related amide-substituted mustard SN 27217 binds in a broadly similar fashion, but with the larger amide group substituent able to reach and contact the side chain of Arg107, further restricting the prodrug conformations in the binding site. The inhibitor dicoumarol appears to bind primarily by pi-stacking interactions and hydrophobic contacts, with no conformational changes in the enzyme. One of the hydroxycoumarin subunits stacks above the plane of the FMN via pi-overlap with the isoalloxazine ring, penetrating deep into the groove, with the other less well-defined. These studies suggest guidelines for further prodrug design. Steric bulk (e.g., mustard rather than aziridine) on the ring can limit the possible binding orientations, and the reducible nitro group must be located para to the mustard. Substitution on the carboxamide side chain still allows the prodrugs to bind, but also limits their orientation in the binding site. Finally, modulating substrate specificity by alteration of the structure of the enzyme rather than the prodrug might usefully focus on modifying the Phe124 residue and those surrounding it.
Annotations from the GeneOntology database. Only terms that fit at least two of the interacting proteins are shown. Molecular function:
6,7-dihydropteridine reductase activity 6,7-dihydropteridine reductase activity
FMN binding FMN binding
identical protein binding identical protein binding
NAD(P)H dehydrogenase (quinone) activity NAD(P)H dehydrogenase (quinone) activity
oxidoreductase activity, acting on other nitrogenous compounds as donors, with NAD or NADP as acceptor oxidoreductase activity, acting on other nitrogenous compounds as donors, with NAD or NADP as acceptor
protein homodimerization activity protein homodimerization activity
Biological process:
2,4,6-trinitrotoluene catabolic process 2,4,6-trinitrotoluene catabolic process
Cellular component:
cytosol cytosol
membrane membrane
Structural annotations of the participating protein chains.Entry contents: 2 distinct polypeptide molecules
Chains: A, B
Notes: All chains according to the most probable oligomerization state stored in PDBe were considered.
Number of unique protein segments: 1
Name: Oxygen-insensitive NAD(P)H nitroreductase
Source organism: Escherichia coli
Length: 217 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMDIISVALKRHSTKAFDASKKLTPEQAEQIKTLLQYSPSSTNSQPWHFIVASTEEGKARVAKSAAGNYVFNERKMLDASHVVVFCAKTAMDDVWLKLVVDQEDADGRFATPEAKAANDKGRKFFADMHRKDLHDDAEWMAKQVYLNVGNFLLGVAALGLDAVPIEGFDAAILDAEFGLKEKGYTSLVVVPVGHHSVEDFNATLPKSRLPQNITLTEV
UniProtKB AC: P38489 (positions: 1-217)
Coverage: 100%
Name: Oxygen-insensitive NAD(P)H nitroreductase
Source organism: Escherichia coli
Length: 217 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMDIISVALKRHSTKAFDASKKLTPEQAEQIKTLLQYSPSSTNSQPWHFIVASTEEGKARVAKSAAGNYVFNERKMLDASHVVVFCAKTAMDDVWLKLVVDQEDADGRFATPEAKAANDKGRKFFADMHRKDLHDDAEWMAKQVYLNVGNFLLGVAALGLDAVPIEGFDAAILDAEFGLKEKGYTSLVVVPVGHHSVEDFNATLPKSRLPQNITLTEV
UniProtKB AC: P38489 (positions: 2-217)
Coverage: 99%
Evidence demonstrating that the participating proteins are unstructured prior to the interaction and their folding is coupled to binding. Representative domain in related structures: Nitroreductase family
Evidence level: Indirect evidence
Evidence coverage: The full structure participates in mutual synergistic folding.
Complex Evidence:
Authors claim that the homodimeric NAD(P)H nitroreductase is a highly intertwined dimer with the FMN binding site lying at the dimer interface (PMID:18241886). Other structures belonging to the nitroreductase family also have an extensive interaction surface wherein a large hydrophobic solvent-accessible surface becomes buried upon dimer formation, suggesting that the monomers would be unstable on their own (PMID:16229462, PMID:19436071). Domain-swapping is also typical, where the extended C-terminal region extensively interacts with the core domain of the neighbouring monomer, forming an interlocked dimer (PMID:34473996, PMID:19436071, PMID:8885832).
Chain A:
N/A
Chain B:
N/A
Surface and contacts features:
Structures from the PDB that contain the same number of proteins, and the proteins from the two structures show a sufficient degree of pairwise similarity, i.e. they belong to the same UniRef90 cluster (the full proteins exhibit at least 90% sequence identity) and convey roughly the same region to their respective interactions (the two regions from the two proteins share a minimum of 70% overlap). Download the CIF file (.cif)
Download this entry's XML file (.xml)
Download this entry's JSON file (.json)
