Protein Engineering 1995-08-01

Enzyme-catalyzed dehalogenation of pentachloroethane: why F87W-cytochrome P450cam is faster than wild type.

J I Manchester, R L Ornstein

Index: Protein Eng. 8(8) , 801-7, (1995)

Full Text: HTML

Abstract

Under anaerobic conditions, cytochromes P450 can reductively dehalogenate heavily halogenated hydrocarbons, such as one- and two-carbon organic solvents. This catalytic capacity has drawn attention to the potential use of engineered forms of P450s in the remediation of contaminated deep subsurface ecosystems. Loida (1994, PhD Thesis, University of Illinois at Urbana-Champaign, IL) and S.G. Sligar (personal communication) have observed recently that an active-site variant of cytochrome P450cam (F87W) dechlorinates pentachloroethane approximately three times faster than the wild-type enzyme. Molecular dynamics simulations have revealed that the mutant enzyme binding pocket remains smaller, and that pentachloroethane assumes configurations closer to the heme-Fe in the F87W mutant twice as often as in the wild-type enzyme. This result is consistent with a collisional model of dehalogenation, which agrees with experimental observations [Li and Wackett (1993) Biochemistry, 32, 9355-9361] that solutions containing wild-type P450cam dehalogenate pentachloroethane 100 times faster than those containing free heme. The simulations suggest that it is unlikely that Trp87 significantly stabilizes the developing negative charge on the substrate during carbon-halogen bond reduction. The design of improved microbial enzymes that incorporate both steric and electronic effects continues for use in remediating halogenated contaminants in situ.