Oxidation of polychlorinated benzenes by genetically engineered CYP101 (cytochrome P450_cam)

Abstract

Polychlorinated benzenes are recalcitrant environmental pollutants primarily because they are resistant to attack by dioxygenases commonly used by micro-organisms for the biodegradation of aromatic compounds. We have investigated the oxidation of polychlorinated benzenes by mutants of the haem mono-oxygenase CYP101 (cytochrome P450 cam ) from Pseudomonas putida with the aim of generating novel systems for their biodegradation. Wildtype CYP101 had low activity for the oxidation of dichlorobenzenes and trichlorobenzenes to the chlorophenols, but no products were detected for the heavily chlorinated benzenes. Increasing the active-site hydrophobicity with the Y96F mutation increased the activity up to 100-fold, and both pentachlorobenzene and hexachlorobenzene were oxidized slowly to pentachlorophenol. Decreasing the space available at the top of the active site with the F87W mutation to force the substrate to be bound closer to the haem resulted in a further 10-fold increase in activity with most substrates. Introducing the F98W mutation, also at the top of the active site, decreased the NADH-turnover rates but increased the coupling efficiencies, and . 90% coupling was observed for 1,3-dichlorobenzene and 1,3,5-trichlorobenzene with the F87W±Y96F± F98W mutant. The V247L mutation generally increased the NADH-turnover rates, and the F87W±Y96F±V247L mutant showed reasonably fast NADH turnover (229 min 21 ) with the highly insoluble pentachlorobenzene without the need for surfactants or organic cosolvents. As all chlorophenols are degraded by micro-organisms, novel biodegradation systems could be constructed in which CYP101 mutants convert the inert polychlorinated benzenes to the phenols, which are then readily degraded by natural pathways.

Crystal Structure of the F87W/Y96F/V247L Mutant of Cytochrome P-450cam with 1,3,5-Trichlorobenzene Bound and Further Protein Engineering for the Oxidation of Pentachlorobenzene and Hexachlorobenzene

“…For convenience, the C334A base mutant is referred to as "wild type," and the Y96F/C334A double mutant is referred to as Y96F, etc. The mutants Y96F, F87W/ Y96F, and F87W/Y96F/V247L had been prepared previously (21). The following oligonucleotides (and their reverse complements) were used on the F87W/Y96F mutant gene to generate the new mutants (target codons underlined): F87W/Y96F/L244A, 5Ј-AAGAGGATGTGTGGCGC-GTTACTGGTCGGCGGC-3Ј; F87W/Y96F/V247A, 5Ј-TGTGGCCTGTT-ACTGGCGGGCGGCCTGGATACG-3Ј; and F87W/Y96F/L244A/V247L, 5Ј-AGGATGTGTGGCGCGTTACTGCTCGGCGGCCTGGAT-3Ј.…”

“…The reactive chlorohydroquinones are then dehalogenated by hydrolytic or reductive mechanisms. We have proposed that chlorophenol degrading micro-organisms could be genetically augmented with monooxygenase enzymes that can convert chlorinated benzenes to the phenol derivatives (20,21). Provided that the introduced enzyme could oxidize the heavily chlorinated benzenes to the phenol derivatives at significant rates, the novel micro-organisms thus generated could degrade all chlorinated benzenes (Fig.…”

“…The We showed recently that the heme monooxygenase CYP101 from Pseudomonas putida (22,23) could be engineered to oxidize all of the lightly chlorinated benzenes with high activity and coupling efficiency compared with the wild type enzyme (20,21). The triple mutant F87W/Y96F/V247L oxidized 1,3,5-trichlororbenzene (1,3,5-TCB) with a catalytic turnover rate of 175 min Ϫ1 and 57% coupling; this activity is more than sufficient for the application of this mutant in the bioremediation of the recalcitrant 1,3,5-TCB.…”

“…The triple mutant F87W/Y96F/V247L oxidized 1,3,5-trichlororbenzene (1,3,5-TCB) with a catalytic turnover rate of 175 min Ϫ1 and 57% coupling; this activity is more than sufficient for the application of this mutant in the bioremediation of the recalcitrant 1,3,5-TCB. This mutant also showed a fast NADH oxidation rate with PeCB as the substrate, but the PeCB oxidation rate was slow because of extensive uncoupling (21). Nevertheless, we were encouraged by the NADH turnover rate because this indicated that the PeCB was bound within the CYP101 active site, although not in the correct orientation to suppress uncoupling side reactions.…”

The ways to increase efficiency of soil bioremediation

“…Lipase activity is responsible for the drastic reduction of total hydrocarbon in the contaminated soil. Research undertaken in this area is likely to contribute to the knowledge on the bioremediation of oil spills [34,46].…”