Issue 4, 2022

Biochemical and structural characterization of Haemophilus influenzae nitroreductase in metabolizing nitroimidazoles

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.

Graphical abstract: Biochemical and structural characterization of Haemophilus influenzae nitroreductase in metabolizing nitroimidazoles

Supplementary files

Article information

Article type
Paper
Submitted
10 dec 2021
Accepted
15 feb 2022
First published
16 feb 2022
This article is Open Access
Creative Commons BY-NC license

RSC Chem. Biol., 2022,3, 436-446

Biochemical and structural characterization of Haemophilus influenzae nitroreductase in metabolizing nitroimidazoles

D. Liu, T. N. Wanniarachchi, G. Jiang, G. Seabra, S. Cao, S. D. Bruner and Y. Ding, RSC Chem. Biol., 2022, 3, 436 DOI: 10.1039/D1CB00238D

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