Nitroheterocycle antibiotics, particularly 5-nitroimidazoles, are frequently used for treating infections caused by anaerobic pathogens. The antimicrobial activities of these drugs heavily rely on the in vivo bioactivation, mainly mediated by widely distributed bacterial nitroreductases. However, the bioactivation can also lead to severe toxicities and drug resistance. In this regard, mechanistic understanding of nitroreductase-mediated 5-nitroimidazole metabolism is essential to address these issues. Here, we report novel metabolism of 5-nitroimidazoles by a human pathogen nitroreductase. Our detailed bioinformatic analysis of nitroreductase homologs uncovered a less-explored, oxygen-insensitive subfamily. We then selected the enzyme from a human pathogen <i>Haemophilus influenzae</i> (HiNfsB) for further studies. Our biochemical characterization of the recombinant enzyme revealed that HiNfsB metabolizes six clinical 5-nitroimidazole antibiotics as well as three nitro-containing anti-TB and immunosuppressive drugs. Notably, three 5-nitroimidazole antibiotics were metabolized in a noncanonical way to produce stable, dimerized products, whose structures were determined by high resolution MS/MS and NMR analysis. Furthermore, X-ray structural analysis of the enzyme coupled with site-directed mutagenesis identified several key residues important to its catalysis and broad substrate scope. Finally, transient expression of HiNfsB in <i>E. coli</i> sensitized the strain to otherwise-resistant 5-nitroimidazoles under anaerobic conditions. Our results discovered the role and mechanistic base of this pathogen nitroreductase in metabolizing 5-nitroimidazoles and other nitro-containing drugs. The new metabolic pathway of 5-nitroimidazoles mediated by HiNfsB advances our fundamental understanding of the metabolism, resistance and toxicity of an important family of antibiotics, guiding their future uses and development.

Novel metabolism of 5-nitroimidazole antibiotics by a human pathogen nitroreductase

ACS Spring 2022

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