Crystal structure of hydroxyquinol 1,2-dioxygenase PnpC from Pseudomonas putida DLL-E4 and its role of N-terminal domain for catalysis.

Hydroxyquinol 1,2-dioxygenase is a key enzyme in the hydroxyquinol pathway of p-nitrophenol (PNP) degradation, and catalyzes the ring cleavage of benzenetriol to maleylacetate. Here, we report the first structure of a hydroxyquinol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida DLL-E4 (PnpC) at the resolution of 2.1 Å. The tertiary structure of PnpC resembles that of the homologous intradiol dioxygenases. The catalytic Fe(III) is pentacoordinated by the conserved Tyr160, Tyr194, His218 and His220, the citrate anion and one water molecule. Among the residues expected to interact with the substrate, structural comparison with the (chloro)catechol dioxygenases suggested that Asp80, Thr81 and Val248 are responsible for the substrate specificity. Moreover, truncation of the N-terminal α-helix of PnpC suggested the N-terminal domain is required for its soluble expression and enzyme catalysis. Our results might provide insights in the substrate recognition and rational design of this enzyme class to be used in bioremediation.