Fig. 3

Biochemical and structural analysis of two-component p-nitrophenol monooxygenase RsNcpAB from Rhodococcus sp. 21391. a Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis of the two components of p-nitrophenol monooxygenase, RsNcpA and RsNcpB. Lane M, protein molecular weight marker. Lane 1, purity of the oxidative component RsNcpA. Lane 2, purity of the oxidative component RsNcpB. b Size-exclusion chromatography of the two-component p-nitrophenol oxygenase using Superdex 200 increase 10/300 GL column. Oligomeric analysis of purified RsNcpA (upper), RsNcpB (middle), and their equimolar mixture (lower) revealed no significant binding between the two components. c Effect of different ratios of A and B proteins on enzyme activity. The final concentration of RsNpcA in the reaction solution was kept at 1 μM, and the enzyme activity was examined with different amounts of RsNpcB. d Overlay of crystal structures of RsNcpA and ligand-bound HpaB (PDB entry: 2YYJ) reveal putative substrate binding sites. e Close-up view of the substrate binding sites. The superimposition of the binding sites of RsNcpA, HpaB, and TftD (PDB entry: 4G5E). f Proposed catalytic mechanism of RsNcpA based on the structural and biochemical analyses. The residue His1293 abstracts a proton from the hydroxyl group of PNP, and a hydroxyl group of the C4a-hydroperoxyflavin intermediate is introduced to the ortho position on the aromatic ring by electrophilic attack. The dienone form of the product is finally rearomatized to p-nitrocatechol, and C4a-hydroxyflavin is formed