Purification and characterization of a benzo[a]pyrene hydroxylase from Pleurotus pulmonarius.
Cytochrome P450 has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by white-rot fungi. We have purified and reconstituted a benzo[a]pyrene hydroxylating cytochrome P450 (P450) from microsomal fractions of the white rot fungus Pleurotus pulmonarius. The microsomal P450 was recovered using a combination of n-aminooctyl agarose and hydroxyapatite chromatography and had an apparent molecular mass of 55 kDa. The purified protein exhibited moderate affinity for benzo[a]pyrene with a K(s) of 66 microM calculated from the Type I substrate binding spectra produced. Reconstitution of activity was achieved and a turnover of 0.75 nmol 3-hydroxybenzo[a]pyrene product/min/nmol P450 was observed, comparable to levels of metabolism observed by animal cytochromes P450 involved in xenobiotic detoxification.
Maspahy
S
S
Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Ceredigion, Wales, SY23 3DA, United Kingdom.
Lamb
D C
DC
Kelly
S L
SL
D001564:Benzo(a)pyrene / Q000378:metabolism; D001579:Benzopyrene Hydroxylase / Q000737:chemistry* / Q000302:isolation & purification; D002248:Carbon Monoxide / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D020076:Pleurotus / Q000201:enzymology*; D011485:Protein Binding; D013053:Spectrophotometry; D013379:Substrate Specificity
D005656:Fungal Proteins; D001564:Benzo(a)pyrene; D009249:NADP; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D001579:Benzopyrene Hydroxylase