A kinetic analysis of three modified novel nitroreductases

A kinetic comparison between three nitroreductase enzymes isolated from the genome of Bacillus licheniformis ATCC 14580 for prospective use as immobilised enzymes for explosives detection has been conducted. The genes encoding the three enzymes (yfkO [BLNfnB] encoding an NfsB-like enzyme; nfrA [BLNfrA1] and ycnD [BLNfrA2] encoding PnrA-like enzymes) have been PCR amplified from the native genome and cloned into pET-28a(+) and a modified cysteine₍₆₎-tagged pET-28a(+) and subsequently over-expressed, purified, and biochemically characterised. The previously uncharacterised nitroreductases exhibited activity against a wide range of explosives, including cyclic nitramines. Amino acid alignments and overall structural comparisons with other nitroreductase family members suggest that the B. licheniformis enzymes are members of the NfsA-Frp/NfsB-FRase I family group. Despite the overall low amino acid identity, regions for flavin mononucleotide binding and active site residues were highly conserved.