The enzymes BaNTR1 (Bacillus amyloliquefaciens, UniProt accession number: A0AAW3IFH5), NfsA (Escherichia coli, UniProt accession number: P17117), BmoNR (Bacillus mojavensis, UniProt accession number: A0AAP3FXD2), and BpNR (Blautia producta, UniProt accession number: A0A7G5MPE8) were expressed as described previously. 21 E. coli BL21(DE3) cells harboring the pET21a plasmid encoding the nitroreductase gene were grown in 1 L of 2xYT medium supplemented with 100 µg/mL ampicillin at 37 °C. Upon reaching an OD600 of 0.6-0.8, protein expression was induced with 0.3 mM IPTG, and the culture was incubated overnight at 18 °C. Cells were harvested by centrifugation, resuspended, and lysed by sonication. The resulting clarified cell lysate (CFL) was incubated with Ni-NTA agarose beads for 3 h at 4 °C with gentle rotation. The mixture was then loaded onto a gravity flow column, and the bound protein was eluted using appropriate bubers. To replenish any FMN cofactor lost during purification, the eluted enzyme was incubated with 1 mM FMN for 3 h at 4 °C with gentle rotation. Finally, the purified enzyme was desalted and buber-exchanged using PD-10 desalting columns (GE Healthcare).