Protein engineering of Bacillus megaterium CYP102 - The oxidation of polycyclic aromatic hydrocarbons

Cytochrome P450 (CYP) enzymes are involved in activating the carcinogenicit y of polycyclic aromatic hydrocarbons (PAHs) in mammals, but they are also utilized by microorganisms for the degradation of these hazardous environme ntal contaminants. Wild-type CYP102 (P450(BM-3)) from Bacillus megaterium h as low activity for the oxidation of the PAHs phenanthrene, fluoranthene an d pyrene. The double hydrophobic substitution R47L/Y51F at the entrance of the substrate access channel increased the PAH oxidation activity by up to 40-fold. Combining these mutations with the active site mutations F87A and A264G lead to order of magnitude increases in activity. Both these mutation s increased the NADPH turnover rate, but the A264G mutation increased the c oupling efficiency while the F87A mutation had dominant effects in product selectivity. Fast NADPH oxidation rates were observed (2250 min(-1) for the R47L/Y51F/F87A mutant with phenanthrene) but the coupling efficiencies wer e relatively low (< 13%), resulting in a highest substrate oxidation rate o f 110 min(-1) for fluoranthene oxidation by the R47L/Y51F/A264G mutant. Mut ation of M354 and L437 inside the substrate access channel reduced PAH oxid ation activity. The PAHs were oxidized to a mixture of phenols and quinones . Notably mutants containing the A264G mutation showed some similarity to m ammalian CYP enzymes in that some 9,10-phenanthrenequinone, the K-region ox idation product from phenanthrene, was formed. The results suggest that CYP 102 mutants could be useful models for PAH oxidation by mammalian CYP enzym es, and also potentially for the preparation of novel PAH bioremediation sy stems.