Database accession: MF7000542
Name: NfsB-T41Q/N71S/F124T mutant (Escherichia coli)
PDB ID: 8c5e
Experimental method: X-ray (1.65 Å)
Assembly: Homodimer
Source organism: Escherichia coli
Primary publication of the structure:
Day MA, Christofferson AJ, Anderson JLR, Vass SO, Evans A, Searle PF, White SA, Hyde EI
Structure and Dynamics of Three NfsB Nitro-Reductase Mutants Selected for Enhanced Activity with the Cancer Prodrug CB1954.
(2023) Int J Mol Sci 24:
PMID: 36983061
Abstract:
Escherichia coli NfsB has been studied extensively for its potential for cancer gene therapy by reducing the prodrug CB1954 to a cytotoxic derivative. We have previously made several mutants with enhanced activity for the prodrug and characterised their activity in vitro and in vivo. Here, we determine the X-ray structure of our most active triple and double mutants to date, T41Q/N71S/F124T and T41L/N71S. The two mutant proteins have lower redox potentials than wild-type NfsB, and the mutations have lowered activity with NADH so that, in contrast to the wild-type enzyme, the reduction of the enzyme by NADH, rather than the reaction with CB1954, has a slower maximum rate. The structure of the triple mutant shows the interaction between Q41 and T124, explaining the synergy between these two mutations. Based on these structures, we selected mutants with even higher activity. The most active one contains T41Q/N71S/F124T/M127V, in which the additional M127V mutation enlarges a small channel to the active site. Molecular dynamics simulations show that the mutations or reduction of the FMN cofactors of the protein has little effect on its dynamics and that the largest backbone fluctuations occur at residues that flank the active site, contributing towards its broad substrate range.
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: 2-217)
Coverage: 99%
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)
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