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 Km and 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 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.
Balashova
N V
NV
Pushchino State University, Moscow region, Russia.
Stolz
A
A
Knackmuss
H J
HJ
Kosheleva
I A
IA
Naumov
A V
AV
Boronin
A M
AM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis; D001673:Biodegradation, Environmental; D002384:Catalysis; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism*; D009284:Naphthols / Q000378:metabolism*; D010616:Phenanthrenes / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003470:Culture Media; D009281:Naphthalenes; D009284:Naphthols; D010616:Phenanthrenes; D009243:NAD; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase; C050175:1-hydroxy-2-naphthoic acid