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 |
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Authors: | NV Balashova A Stolz H-J Knackmuss IA Kosheleva AV Naumov AM Boronin |
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Institution: | (1) Pushchino State University, 142290, Pushchino, Moscow region, Russia;(2) Institut für Mikrobiologie der Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany;(3) Laboratory of Plasmid Biology, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290, Pushchino Moscow region, Russia Author for correspondence: tel |
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Abstract: | 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-naphthoateto 1,2-dihydroxynaphthalene was purified from the phenanthrene- and naphthalene-degrading Pseudomonas putida strain BS202-P1. The purified protein had a molecular weight of45 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 Kmand Vmax 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 salicylate1-monooxygenase. Together with previous studies, this suggested that the enzymes of the naphthalene pathway are sufficient to catalyse also the mineralization of phenanthrene. |
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Keywords: | 1-Hydroxy-2-naphthoate naphthalene phenanthrene Pseudomonas putida salicylate hydroxylase |
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