Purification and Characterization of a Benzo[a]pyrene Hydroxylase from Pleurotus pulmonarius

• Published in: Biochemical and biophysical research communications 1999-12, Vol.266 (2), p.326-329
• Main Authors: Maspahy, Segula, Lamb, David C., Kelly, Steven L.
• Format: Article
• Language: English
• Subjects: Benzo(a)pyrene - metabolism; benzo[@@ia@]pyrene hydroxylase; Benzopyrene Hydroxylase - chemistry; Benzopyrene Hydroxylase - isolation & purification; bioremediation; biotransformation; Carbon Monoxide - pharmacology; Cytochrome P-450 Enzyme System - chemistry; Fungal Proteins - chemistry; Kinetics; Microsomes - enzymology; NADP - metabolism; Pleurotus - enzymology; Pleurotus pulmonarius; pollutants; Protein Binding; Spectrophotometry; Substrate Specificity
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1999.1813

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 Ks of 66 μM 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.