Fig. 5. Proposed dimeric metabolites generated from nitroimidazoles by HiNfsB. (A) LC traces of the HiNfsB reactions with compounds 5, 6 and 7 as substrates. The 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. Negative control (NC) lacked enzyme. The peaks of substrates and new metabolites are highlighted in grey and orange, respectively. HRMS spectra of these metabolites were included along with their putative chemical structures. (B) EIC analysis (m/z = 431.15) identified the putative dimeric metabolite from the crude extract of HiNfsB expressing E. coli culture fed with 6 (100 μM). (i) E. coli with the empty vector pET22b; (ii) EcNfsB expressing E. coli; (iii) HiNfsB expressing E. coli; (iv): HiNfsB expressing E. coli induced with 0.1 μM IPTG. All cultures were fed with 100 μM 6 and then incubated at 250 rpm, 37 °C overnight under aerobic conditions. Putative dimeric product was highlighted in orange.