Fig. 3. Recombinant HiNfsB metabolizes selected nitroimidazoles. (A) LC traces of the HiNfsB reactions with metronidazole (2) as substrate. The reactions contained 2 mM substrate, 1 μM enzyme, 1 mM NADP⁺, 20 mM glucose, and 16 μM glucose dehydrogenase (GDH) in 100 mM phosphate buffer (pH 7.4) and were incubated at room temperature for 3 hours under aerobic (i) or anaerobic (ii) condition. Negative controls lacked NADP⁺ (iii) or enzyme (iv). MTZ peak is highlighted in grey. (B) Consumption of 14 nitro-containing compounds by HiNfsB with or without 1 μM supplemented FMN. The reaction conditions were the same as (A) expect for the use of 0.1 μM enzyme and incubation for 10 min. The substrate was detected at 320 nm or 276 nm (for azathioprine). Consumptions (%) were determined as substrate peak areas at 10 min per those at 0 min × 100. The data represent means ± s.d. of three independent experiments.