Purification of cytochrome P450 and ferredoxin, involved in bisphenol A degradation, from Sphingomonas sp. strain AO1.
In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450bisd), ferredoxin (Fd(bisd)), and ferredoxin reductase (Red(bisd)). We demonstrated that P450bisd and Fd(bisd) are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd(bisd) revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450(bisd), in the presence of Fd(bisd), Red(bisd), and NADH, was able to convert BPA. The K(m) and kcat values for BPA degradation were 85 +/- 4.7 microM and 3.9 +/- 0.04 min(-1), respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450bisd might exhibit strict specificity. Two BPA degradation products of the P450(bisd) system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.
Sasaki
Miho
M
Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan.
Akahira
Ayako
A
Oshiman
Ko-ichi
K
Tsuchido
Tetsuaki
T
Matsumura
Yoshinobu
Y
D000395:Air Pollutants, Occupational; D000595:Amino Acid Sequence; D001559:Benzhydryl Compounds; D001673:Biodegradation, Environmental; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005288:Ferredoxins / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010636:Phenols / Q000493:pharmacokinetics*; D020578:Sphingomonas / Q000201:enzymology / Q000378:metabolism*; D000098826:Bisphenol A Compounds
D000395:Air Pollutants, Occupational; D001559:Benzhydryl Compounds; D003577:Cytochrome P-450 Enzyme System; D005288:Ferredoxins; D010446:Peptide Fragments; D010636:Phenols; C006780:bisphenol A; D000098826:Bisphenol A Compounds