Database accession: MF7000539
Name: Nitroreductase with nicotinic acid (Haemophilus influenzae Rd KW20)
PDB ID: 7t33
Experimental method: X-ray (2.30 Å)
Assembly: Homodimer
Source organism: Haemophilus influenzae
Primary publication of the structure:
Liu D, Wanniarachchi TN, Jiang G, Seabra G, Cao S, Bruner SD, Ding Y
Biochemical and structural characterization of nitroreductase in metabolizing nitroimidazoles.
(2022) Rsc Chem Biol 3: 436-446
PMID: 35441146
Abstract:
Nitroheterocycle antibiotics, particularly 5-nitroimidazoles, are frequently used for treating anaerobic infections. The antimicrobial activities of these drugs heavily rely on the in vivo bioactivation, mainly mediated by widely distributed bacterial nitroreductases (NTRs). However, the bioactivation can also lead to severe toxicities and drug resistance. Mechanistic understanding of NTR-mediated 5-nitroimidazole metabolism can potentially aid addressing these issues. Here, we report the metabolism of structurally diverse nitroimidazole drug molecules by a NTR from a human pathogen Haemophilus influenzae (HiNfsB). Our detailed bioinformatic analysis uncovered that HiNfsB represents a group of unexplored oxygen-insensitive NTRs. Biochemical characterization of the recombinant enzyme revealed that HiNfsB effectively metabolizes ten clinically used nitroimidazoles. Furthermore, HiNfsB generated not only canonical nitroreduction metabolites but also stable, novel dimeric products from three nitroimidazoles, whose structures were proposed based on the results of high resolution MS and tandem MS analysis. X-ray structural analysis of the enzyme coupled with site-directed mutagenesis identified four active site residues important to its catalysis and broad substrate scope. Finally, transient expression of HiNfsB sensitized an E. coli mutant strain to 5-nitroimidazoles under anaerobic conditions. Together, these results advance our understanding of the metabolism of nitroimidazole antibiotics mediated by a new NTR group and reinforce the research on the natural antibiotic resistome for addressing the antibiotic resistance crisis.
Annotations from the GeneOntology database. Only terms that fit at least two of the interacting proteins are shown. Molecular function:
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
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, B
Notes: All chains according to the most probable oligomerization state stored in PDBe were considered.
Number of unique protein segments: 1
Name: Putative NAD(P)H nitroreductase
Source organism: Haemophilus influenzae
Length: 220 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMTQLTREQVLELFHQRSSTRYYDPTKKISDEDFECILECGRLSPSSVGSEPWKFLVIQNKTLREKMKPFSWGMINQLDNCSHLVVILAKKNARYDSPFFVDVMARKGLNAEQQQAALTKYKALQEEDMKLLENDRTLFDWCSKQTYIALANMLTGASALGIDSCPIEGFHYDKMNECLAEEGLFDPQEYAVSVAATFGYRSRDIAKKSRKGLDEVVKWVG
UniProtKB AC: Q57431 (positions: 2-220)
Coverage: 99%
Name: Putative NAD(P)H nitroreductase
Source organism: Haemophilus influenzae
Length: 220 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMTQLTREQVLELFHQRSSTRYYDPTKKISDEDFECILECGRLSPSSVGSEPWKFLVIQNKTLREKMKPFSWGMINQLDNCSHLVVILAKKNARYDSPFFVDVMARKGLNAEQQQAALTKYKALQEEDMKLLENDRTLFDWCSKQTYIALANMLTGASALGIDSCPIEGFHYDKMNECLAEEGLFDPQEYAVSVAATFGYRSRDIAKKSRKGLDEVVKWVG
UniProtKB AC: Q57431 (positions: 2-220)
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)
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