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-r ot fungi. We have purified and reconstituted a benzo[a]pyrene hydroxylating cytochrome P450 (P450) from microsomal fractions of the white rot fungus P leurotus pulmonarius. The microsomal P450 was recovered using a combination of n-aminooctyl agarose and hydroxyapatite chromatography and had an appar ent molecular mass of 55 kDa. The purified protein exhibited moderate affin ity for benzo[a]pyrene with a K-s of 66 mu M calculated from the Type I sub strate binding spectra produced. Reconstitution of activity was achieved an d a turnover of 0.75 nmol 3-hydroxy-benzo[a]pyrene product/min/nmol P450 wa s observed, comparable to levels of metabolism observed by animal cytochrom es P450 involved in xenobiotic detoxification. (C) 1999 Academic Press.