Fig. 4. Metabolites produced from nitroimidazoles by HiNfsB. (A–C) EIC analysis of hydroxylamine and amine metabolites in the HiNfsB reactions with compounds 8, 10 and 11 as substrates, respectively. The full reactions contained 2 mM substrate, 1 μM enzyme, 1 mM NADP⁺, 20 mM glucose, and 16 μM GDH in 100 mM phosphate buffer (pH 7.4) and were incubated at room temperature for 3 hours. Corresponding m/z values were shown. (D–F) EIC analysis of CBZ derivatives in the HiNfsB reactions with compounds 4, 5 and 7 as substrates, respectively. The reaction conditions are the same as above, except for the use of 2 μM enzyme and incubation for 5 min. Negative control (NC) lacked enzyme. Corresponding m/z values were shown. The peaks of hydroxylamine metabolites and their CBZ derivatives are highlighted in grey, while those of amine metabolites and their CBZ derivatives are highlighted in orange.