Database accession: MF7000545
Name: NfsB_Vv F70A/F108Ywith FMN (Vibrio vulnificus)
PDB ID: 7uwt
Experimental method: X-ray (1.85 Å)
Assembly: Homodimer
Source organism: Vibrio vulnificus
Primary publication of the structure:
Sharrock AV, Mumm JS, Bagdžiūnas G, Čėnas N, Arcus VL, Ackerley DF
The Crystal Structure of Engineered Nitroreductase NTR 2.0 and Impact of F70A and F108Y Substitutions on Substrate Specificity.
(2023) Int J Mol Sci 24:
PMID: 37047605
Abstract:
Bacterial nitroreductase enzymes that convert prodrugs to cytotoxins are valuable tools for creating transgenic targeted ablation models to study cellular function and cell-specific regeneration paradigms. We recently engineered a nitroreductase ("NTR 2.0") for substantially enhanced reduction of the prodrug metronidazole, which permits faster cell ablation kinetics, cleaner interrogations of cell function, ablation of previously recalcitrant cell types, and extended ablation paradigms useful for modelling chronic diseases. To provide insight into the enhanced enzymatic mechanism of NTR 2.0, we have solved the X-ray crystal structure at 1.85 Angstroms resolution and compared it to the parental enzyme, NfsB from Vibrio vulnificus. We additionally present a survey of reductive activity with eight alternative nitroaromatic substrates, to provide access to alternative ablation prodrugs, and explore applications such as remediation of dinitrotoluene pollutants. The predicted binding modes of four key substrates were investigated using molecular modelling.
Annotations from the GeneOntology database. Only terms that fit at least two of the interacting proteins are shown. Molecular function:
nucleotide binding nucleotide binding
oxidoreductase activity oxidoreductase activity
Biological process:
2,4,6-trinitrotoluene catabolic process 2,4,6-trinitrotoluene catabolic process
Cellular component:
cytosol cytosol
Structural annotations of the participating protein chains.Entry contents: 2 distinct polypeptide molecules
Chains: A, A-2
Notes: All chains according to the most probable oligomerization state stored in PDBe were considered.
Number of unique protein segments: 1
Name: Nitroreductase
Source organism: Vibrio vulnificus
Length: 217 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMTIVQAAQSRYSTKAFDASRKLPEEKVAAVKELIRMSASSVNSQPWHFIVASSEEGKARIAKATQGGFAFNERKILDASHVVVFCAKTAIDEAYLLDLLESEDKDGRFADVEAKNGMHAGRSFFVNMHRFDLKDAHHWMEKQVYLNVGTLLLGASAMEIDAVPIEGFDAKVLDEEFGLREKGFTSVVIVPLGYHSEDDFNAKLPKSRWSAETVFTEI
UniProtKB AC: Q7MCD2 (positions: 1-217)
Coverage: 100%
Name: Nitroreductase
Source organism: Vibrio vulnificus
Length: 217 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMTIVQAAQSRYSTKAFDASRKLPEEKVAAVKELIRMSASSVNSQPWHFIVASSEEGKARIAKATQGGFAFNERKILDASHVVVFCAKTAIDEAYLLDLLESEDKDGRFADVEAKNGMHAGRSFFVNMHRFDLKDAHHWMEKQVYLNVGTLLLGASAMEIDAVPIEGFDAKVLDEEFGLREKGFTSVVIVPLGYHSEDDFNAKLPKSRWSAETVFTEI
UniProtKB AC: Q7MCD2 (positions: 1-217)
Coverage: 100%
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 A-2:
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)
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