Database accession: MF7000530
Name: NfsA with hydroquinone (Escherichia coli)
PDB ID: 7nmp
Experimental method: X-ray (1.25 Å)
Assembly: Homodimer
Source organism: Escherichia coli
Primary publication of the structure:
Day MA, Jarrom D, Christofferson AJ, Graziano AE, Anderson JLR, Searle PF, Hyde EI, White SA
The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN.
(2021) Biochem. J. 478: 2601-2617
PMID: 34142705
Abstract:
NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH- to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor.
Annotations from the GeneOntology database. Only terms that fit at least two of the interacting proteins are shown. Molecular function:
chromate reductase activity chromate reductase activity
FMN binding FMN binding
NAD(P)H dehydrogenase (quinone) activity NAD(P)H dehydrogenase (quinone) activity
protein homodimerization activity protein homodimerization activity
Biological process: not assigned
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: Oxygen-insensitive NADPH nitroreductase
Source organism: Escherichia coli
Length: 240 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMTPTIELICGHRSIRHFTDEPISEAQREAIINSARATSSSSFLQCSSIIRITDKALREELVTLTGGQKHVAQAAEFWVFCADFNRHLQICPDAQLGLAEQLLLGVVDTAMMAQNALIAAESLGLGGVYIGGLRNNIEAVTKLLKLPQHVLPLFGLCLGWPADNPDLKPRLPASILVHENSYQPLDKGALAQYDEQLAEYYLTRGSNNRRDTWSDHIRRTIIKESRPFILDYLHKQGWATR
UniProtKB AC: P17117 (positions: 1-240)
Coverage: 100%
Name: Oxygen-insensitive NADPH nitroreductase
Source organism: Escherichia coli
Length: 240 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMTPTIELICGHRSIRHFTDEPISEAQREAIINSARATSSSSFLQCSSIIRITDKALREELVTLTGGQKHVAQAAEFWVFCADFNRHLQICPDAQLGLAEQLLLGVVDTAMMAQNALIAAESLGLGGVYIGGLRNNIEAVTKLLKLPQHVLPLFGLCLGWPADNPDLKPRLPASILVHENSYQPLDKGALAQYDEQLAEYYLTRGSNNRRDTWSDHIRRTIIKESRPFILDYLHKQGWATR
UniProtKB AC: P17117 (positions: 1-240)
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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