Purification and characterization of a salicylate hydroxylase involved in 1-hydroxy-2-naphthoic acid hydroxylation from the naphthalene and phenanthrene-degrading bacterial strain Pseudomonas putida BS202-P1

1-Hydroxy-2-naphthoate is formed as an intermediate in the bacterial degradation of phenanthrene. A monooxygenase which catalyzed the oxidation of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene was purified from the phenanthrene- and naphthalene-degrading Pseudomonas putida strain BS202-P1. The purified protein had a molecular weight of 45 kDa and required NAD(P)H and FAD as cofactors. The purified enzyme also catalysed the oxidation of salicylate and various substituted salicylates. The comparison of the K-m and V-max values for 1-hydroxy-2-naphthoate and salicylate demonstrated a higher catalytic efficiency of the enzyme for salicylate as a substrate. A significant substrate-inhibition was detected with higher concentrations of 1-hydroxy-2-naphthoate. The aminoterminal amino acid sequence of the purified enzyme showed significant homologies to salicylate 1-monooxygenases from other Gram negative bacteria. It was therefore concluded that during the degradation of phenanthrene the conversion of 1-hydroxy-2-naphthoate to 1,2-dihydroxynaphthalene is catalysed by a salicylate 1-monooxygenase. Together with previous studies, this suggested that the enzymes of the naphthalene pathway are sufficient to catalyse also the mineralization of phenanthrene.