Oxidation of polychlorinated benzenes by genetically engineered CYP101 (cytochrome P450(cam))

Polychlorinated benzenes are recalcitrant environmental pollutants primaril y because they are resistant to attack by dioxygenases commonly used by mic ro-organisms for the biodegradation of aromatic compounds. We have investig ated the oxidation of polychlorinated benzenes by mutants of the haem mono- oxygenase CYP101 (cytochrome P450(cam)) from Pseudomonas putida with the ai m of generating novel systems for their biodegradation. Wild-type CYP101 ha d low activity for the oxidation of dichlorobenzenes and trichlorobenzenes to the chlorophenols, but no products were detected for the heavily chlorin ated benzenes. Increasing the active-site hydrophobicity with the Y96F muta tion increased the activity up to 100-fold, and both pentachlorobenzene and hexachlorobenzene were oxidized slowly to pentachlorophenol. Decreasing th e space available at the top of the active site with the F87W mutation to f orce the substrate to be bound closer to the haem resulted in a further 10- fold increase in activity with most substrates. Introducing the F98W mutati on, also at the top of the active site, decreased the NADH-turnover rates b ut increased the coupling efficiencies, and > 90% coupling was observed for 1,3-dichlorobenzene and 1,3,5-trichlorobenzene with the F87W-Y96F-F98W mut ant. The V247L mutation generally increased the NADH-turnover rates, and th e F87W-Y96F-V247L mutant showed reasonably fast NADH turnover (229 min(-1)) with the highly insoluble pentachlorobenzene without the need for surfacta nts or organic cosolvents. As all chlorophenols are degraded by micro-organ isms, novel biodegradation systems could be constructed in which CYP101 mut ants convert the inert polychlorinated benzenes to the phenols, which are t hen readily degraded by natural pathways.