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

Abstract

We reported previously that the F87W/Y96F/V247L mutant of cytochrome P-450cam (CYP101) from Pseudomonas putida catalyzed the rapid oxidation of lightly chlorinated benzenes, but pentachlorobenzene oxidation was slow (Jones, J. P., O'Hare, E. J., and Wong, L. L. (2001) Eur. J. Biochem. 268, 1460 -1467). In the present work, we determined the crystal structure of this mutant with bound 1,3,5-trichlorobenzene. The substrate was bound to crystallographically independent CYP101 molecules in at least three different orientations, which were distinguished by the angle between the benzene ring and the porphyrin, and one orientation contained an Fe-Cl interaction. In another orientation, the substrate was almost parallel to the heme, with a C-H bond closest to the iron. The enzyme/substrate contacts suggested that the L244A mutation should promote the binding of pentachlorobenzene and hexachlorobenzene by creating space to accommodate the extra chlorines. The F87W/Y96F/L244A/V247L mutant thus designed was found to oxidize pentachlorobenzene at a rate of 82.5 nmol (nmol CYP101) ؊1 min ؊1 , 45 times faster than the F87W/Y96F/V247L parent mutant. The rate of hexachlorobenzene oxidation was increased 200-fold, to 2.0 min ؊1 . Both substrates are oxidized to pentachlorophenol, which is degraded by micro-organisms. In principle, the F87W/ Y96F/L244A/V247L mutant could have applications in the bioremediation of polychlorinated benzenes.

Conversion of Sphingobium chlorophenolicum ATCC 39723 to a Hexachlorobenzene Degrader by Metabolic Engineering

“…It had been demonstrated that the F87W/Y96F/L244A/ V247L mutant of cytochrome P-450 cam (CYP101) can oxidize HCB to pentachlorophenol (PCP) (4), which can be completely degraded by many microorganisms in the environment. In particular, the catabolic pathway of PCP in Sphingobium chlorophenolicum ATCC 39723 has been characterized thoroughly both biochemically and genetically (2).…”

Aerobic biodegradation of hexachlorobenzene by an acclimated microbial community

“…No strictly aerobic organism capable of degrading HCB has been isolated (Chen et al, 2002). In this study, we report our investigations on the aerobic biotransformation of HCB, and the effects of different process parameters such as variations in pH values, and temperatures on the degradation efficiency of HCB by an acclimated microbial community -a mixed culture.…”

“…Most studies on HCB degradation have been conducted under anaerobic conditions (Holliger et al, 1992;Nowak et al, 1996;Adrian et al, 1998Adrian et al, , 2000Pavlostathis and Prytula, 2000;Wu et al, 2002), and only few studies have under aerobic biodegradation processes (Chen et al, 2002;Gan et al, 2001). No strictly aerobic organism capable of degrading HCB has been isolated (Chen et al, 2002).…”

The enzymatic basis for pesticide bioremediation

“…One good example is a variant of P450 cam (F87W\ Y96F\L244A\V247L) from Pseudomonas putida that has been demonstrated to have signifi cant activity against the key chlorinated pollutants pentachlorobenzene (a k cat of 82.5 min -1 ) and hexachlorobenzene (a k cat of 2.5 min -1 ) [27]. This variant of the P450 cam enzyme has now been used to endow the capacity to completely degrade hexachlorobenzane upon a Sphingobium chlorophenolicum species [28].…”