Crystal structure of p-nitrophenol 4-monooxygenase PnpA from Pseudomonas putida DLL-E4: The key enzyme involved in p-nitrophenol degradation

p-Nitrophenol 4-monooxygenase PnpA, the key enzyme in the hydroquinone pathway of p-nitrophenol (PNP) degradation, catalyzes the monooxygenase reaction of PNP to p-benzoquinone in the presence of FAD and NADH. Here, we determined the first crystal structure of PnpA from Pseudomonas putida DLL-E4 in its apo and FAD-complex forms to a resolution of 2.04 Å and 2.48 Å, respectively. The PnpA structure shares a common fold with hydroxybenzoate hydroxylases, despite a low amino sequence identity of 14–18%, confirming it to be a member of the Class A flavoprotein monooxygenases. However, substrate docking studies of PnpA indicated that the residues stabilizing the substrate in an orientation suitable for catalysis are not observed in other homologous hydroxybenzoate hydroxylases, suggesting PnpA employs a unique catalytic mechanism. This work expands our understanding on the reaction mode for this enzyme class. •This is the first structure of PnpA determined by X-ray crystallography.•Structure of PnpA is similar to Class A flavoprotein monooxygenases.•The substrate of PnpA is coordinated by cysteine and arginine, which is different from other PHBH family enzymes.