B-256. Kinetic Studies of RdxA and FrxA Nitroreductases Involved in Metronidazole- Sensitivity of Helicobacter pylori

I. N. Olekhnovich, P. S. Hoffman;
Univ. of Virginia, Charlottesville, VA.

Metronidazole (MTZ) is a nitroimidazole prodrug that is widely used in eradication therapy against the gastric pathogen H. pylori. Metronidazole resistance is common and reduces the efficacy of MTZ-containing eradication regimes. Genetic evidence indicates that sequential loss of function mutations in rdxA and frxA oxygen-insensitive NAD(P)H-dependent nitroreductases correlate with high level resistance to MTZ. However, the enzymatic mechanisms of this action remain unclear. Here we report cloning, overexpression, and purification of N-terminal His-tagged versions of RdxA (26.25 KDa) and FrxA (27.4 kDa) proteins from E. coli using Ni-affinity chromatography. The gel filtration experiments determined that both proteins are dimers. Solutions of the RdxA and FrxA proteins were yellow, and the optical spectra obtained for the proteins were consistent with flavin mononucleotide (FMN) as cofactor. Enzymatic analysis performed side by side with NfsB, one of the best characterized NAD(P)H-dependent nitroreductases from E. coli, showed that RdxA and FrxA can use either NADH or NADPH as substrates in reduction of a number of nitro compounds and quinones including: nitrofurazone, nitrofurantoin, furazolidone, and 1,4-benzoquinone. Surprisingly, metronidazole was not a substrate for either RdxA or FrxA nitroreductases in vitro. Kinetic studies of initial velocities of RdxA and FrxA demonstrated that both enzymes employ a ping-pong bi-bi mechanism, as described for other nitroreductases. Our data are consistent with a model in which RdxA and FrxA promote MTZ sensitivity indirectly by contributing to maintenance of a low cytoplasmic redox potential in H. pylori.