Pseudomonas sp. strain PPD can metabolize phenanthrene as the sole source of carbon and energy via the 'phthalic acid' route. The key enzyme, 1-hydroxy-2-naphthoic acid dioxygenase (1-HNDO, EC 1.13.11.38), was purified to homogeneity using a 3-hydroxy-2-naphthoic acid (3-H2NA)-affinity matrix. The enzyme was a homotetramer with a native molecular mass of 160 kDa and subunit molecular mass of ~39 kDa. It required Fe(II) as the cofactor and was specific for 1-hydroxy-2-naphthoic acid (1-H2NA), with Km 13.5 占and Vmax 114?孯l min-1 mg-1. 1-HNDO failed to show activity with gentisic acid, salicylic acid and other hydroxynaphthoic acids tested. Interestingly, the enzyme showed substrate inhibition with a Ki of 116 卮 1-HNDO was found to be competitively inhibited by 3-H2NA with a Ki of 24 卮 Based on the pH-dependent spectral changes, the enzyme reaction product was identified as 2-carboxybenzalpyruvic acid. Under anaerobic conditions, the enzyme failed to convert 1-H2NA to 2-carboxybenzalpyruvic acid. Stoichiometric studies showed the incorporation of 1 mol O2 into the substrate to yield 1 mol product. These results suggest that 1-HNDO from Pseudomonas sp. strain PPD is an extradiol-type ring-cleaving dioxygenase.