Structure of R dx A – an oxygen‐insensitive nitroreductase essential for metronidazole activation in H elicobacter pylori

The R dx A oxygen‐insensitive nitroreductase of the human gastric pathogen H elicobacter pylori is responsible for the susceptibility of this organism to the redox active prodrug metronidazole [2‐(2‐methyl‐5‐nitro‐1 H ‐imidazol‐1‐yl)ethanol]. Loss‐of‐function mutations in rdxA are primarily responsible for resistance to this therapeutic. R dx A exhibits potent NADPH oxidase activity under aerobic conditions and metronidazole reductase activity under strictly anaerobic conditions. In the present study, we report the crystal structure of R dx A , which is a homodimer exhibiting domain swapping and containing two molecules of FMN bound at the dimer interface. We have found a gap between the side chain of T yr47 and the isoalloxazine ring of FMN that appears to be appropriate for substrate binding. The structure does not include residues 97–128, which correspond to a locally unstable part of the NTR from Escherichia coli , and might be involved in cofactor binding. Comparison of H . pylori R dx A with other oxidoreductases of known structure suggests that R dx A may belong to a new subgroup of oxidoreductases in which a cysteine side chain close to the FMN cofactor could be involved in the reductive activity. In this respect, the mutation of C159 to A or S ( C 159 A / S ) has resulted in a loss of metronidazole reductase activity but not NADPH oxidase activity. The R dx A structure enables the interpretation of the many loss‐of‐function mutations described previously, including those affecting C 159, a residue whose interaction with FMN is required for the nitroreduction of metronidazole. The present studies provide unique insights into the redox behaviour of the flavin in this key enzyme for metronidazole activation, including a potential use in gene therapy. Database Structural data have been deposited in the Protein Data Bank under accession number 3QDL .