%0 Journal Article %A Deveryshetty, Jaigeeth %A Phale, Prashant S. %T Biodegradation of phenanthrene by Pseudomonas sp. strain PPD: purification and characterization of 1-hydroxy-2-naphthoic acid dioxygenase %D 2009 %J Microbiology, %V 155 %N 9 %P 3083-3091 %@ 1465-2080 %R https://doi.org/10.1099/mic.0.030460-0 %K 1-H2NA, 1-hydroxy-2-naphthoic acid %K P3,4DO, protocatechuate 3,4-dioxygenase %K SDO, salicylic acid 1,2-dioxygenase %K 1-HNDO, 1-hydroxy-2-naphthoic acid dioxygenase %K 3-H2NA, 3-hydroxy-2-naphthoic acid %K 6-H2NA, 6-hydroxy-2-naphthoic acid %K GDO, gentisic acid 1,2-dioxygenase %K 2-CBADH, 2-carboxybenzaldehyde dehydrogenase %K P4,5DO, protocatechuic acid 4,5-dioxygenase %K 2-H1NA, 2-hydroxy-1-naphthoic acid %I Microbiology Society, %X 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 K m 13.5 μM and V max 114 μmol 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 K i of 116 μM. 1-HNDO was found to be competitively inhibited by 3-H2NA with a K i of 24 μM. 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. %U https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.030460-0