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 mu M calculated from the Type I substrate binding spectra produced. Reconstitution of activity was achieved and a turnover of 0.75 nmol 3-hydroxy-benzo[a]pyrene product/min/nmol P450 was observed, comparable to levels of metabolism observed by animal cytochromes P450 involved in xenobiotic detoxification. (C) 1999 Academic Press.