Biodegradation of phenanthrene by Alcaligenes sp. strain PPH: partial purification and characterization of 1-hydroxy-2-naphthoic acid hydroxylase.
Alcaligenes sp. strain PPH degrades phenanthrene via 1-hydroxy-2-naphthoic acid (1-H2NA), 1,2-dihydroxynaphthalene (1,2-DHN), salicylic acid and catechol. Enzyme activity versus growth profile and heat stability studies suggested the presence of two distinct hydroxylases, namely 1-hydroxy-2-naphthoic acid hydroxylase and salicylate hydroxylase. 1-Hydroxy-2-naphthoic acid hydroxylase was partially purified (yield 48%, fold 81) and found to be a homodimer with a subunit molecular weight of ∼34 kDa. The enzyme was yellow in color, showed UV-visible absorption maxima at 274, 375 and 445 nm, and fluorescence emission maxima at 527 nm suggested it to be a flavoprotein. The apoenzyme prepared by the acid-ammonium sulfate (2 M) dialysis method was colorless, inactive and lost the characteristic flavin absorption spectra but regained ∼90% activity when reconstituted with FAD. Extraction of the prosthetic group and its analysis by HPLC suggests that the holoenzyme contained FAD. The enzyme was specific for 1-H2NA and failed to show activity with any other hydroxynaphthoic acid analogs or salicylic acid. The K(m) for 1-H2NA in the presence of either NADPH or NADH remained unaltered (72 and 75 μM, respectively), suggesting dual specificity for the coenzyme. The K(m) for FAD was determined to be 4.7 μM. The enzyme catalyzed the conversion of 1-H2NA to 1,2-DHN only under aerobic conditions. These results suggested that 1-hydroxy-2-naphthoic acid hydroxylase is a flavoprotein monooxygenase specific for 1-H2NA and different from salicylate-1-hydroxylase.
Deveryshetty
Jaigeeth
J
Department of Biosciences and Bioengineering, Indian Institute Technology Bombay, Mumbai, Maharashtra, India.
Phale
Prashant S
PS
D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001673:Biodegradation, Environmental; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009284:Naphthols / Q000378:metabolism*; D010616:Phenanthrenes / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D009284:Naphthols; D010616:Phenanthrenes; C031181:phenanthrene; D006899:Mixed Function Oxygenases; C050175:1-hydroxy-2-naphthoic acid