Protein engineering of cytochrome P450(cam) (CYP101) for the oxidation of polycyclic aromatic hydrocarbons

Mutations of the active site residues F87 and Y96 greatly enhanced the acti vity of cytochrome P450(cam) (CYP101) from Pseudomonas putida for the oxida tion of the polycyclic aromatic hydrocarbons phenanthrene, fluoranthene, py rene and benzo[a]pyrene. Wild-type P450(cam) had low (<0.01 min(-1)) activi ty with these substrates, Phenanthrene was oxidized to 1-, 2-, 3- and 4-phe nanthrol, while fluoranthene gave mainly 3-fluoranthol, Pyrene was oxidized to 1-pyrenol and then to 1,6- and 1,8-pyrenequinone, with small amounts of 2-pyrenol also formed with the Y96A mutant. Benzo[a]pyrene gave 3-hydroxyb enzo[a]pyrene as the major product. The NADH oxidation rate of the mutants with phenanthrene was as high as 374 min(-1), which was 31% of the camphor oxidation rate by wild-type P450(cam), and with fluoranthene the fastest ra te was 144 min(-1). The oxidation of phenanthrene and fluoranthene were hig hly uncoupled, with highest couplings of 1.3 and 3.1%, respectively. The hi ghest coupling efficiency for pyrene oxidation was a reasonable 23%, but th e NADH turnover rate was slow. The product distributions varied significant ly between mutants, suggesting that substrate binding orientations can be m anipulated by protein engineering, and that genetic variants of P450(cam) m ay be useful for studying the oxidation of polycyclic aromatic hydrocarbons by P450 enzymes.