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  • Title: Distal site and surface mutations of cytochrome P450 1A2 markedly enhance dehalogenation of chlorinated hydrocarbons.
    Author: Yanagita K, Sagami I, Shimizu T.
    Journal: Arch Biochem Biophys; 1997 Oct 15; 346(2):269-76. PubMed ID: 9343374.
    Abstract:
    Chlorinated compounds such as chlorinated ethylenes and ethanes are serious environmental pollutants. In the present study, we examined whether or not a recombinant strain of Saccharomyces cerevisiae that expresses rat liver cytochrome P450 1A2 (P450 1A2) wild-type and mutant proteins can efficiently catalyze oxidative and reductive dehalogenations of trichloroethylene, pentachloroethane, and hexachloroethane. Mutations at putative heme distal and protein surface sites of P450 1A2 greatly enhanced turnover values toward those substrates under both aerobic and anaerobic conditions. For example, a Thr319Ala mutation at the putative heme distal site enhanced the degradation rate of trichloroethylene and pentachloroethane by 2- and 2.7-fold, respectively, under aerobic conditions. The Thr319Ala mutation also strongly facilitated the reaction with hexachloroethane up to 13- and 4.5-fold under aerobic and anaerobic conditions, respectively. The Thr319Ala mutation increased dechlorinated over protonated product ratios by 3-fold as well when either pentachloroethane or hexachloroethane was used as a substrate. A Lys250Leu mutation on the putative protein surface site enhanced the dehalogenation rate of hexachloroethane up to 4.8-fold under anaerobic conditions. In contrast, a Glu318Ala mutation at the putative distal site markedly decreased the activities with trichloroethylene and pentachloroethane substrates under aerobic conditions. Conserved amino acids Thr319 and Glu318 at the heme distal site have been suggested to be important in the O2 activation during monooxidation reactions of P450s. However, the present study indicates that Thr319 is likely to be an inhibitor of dechlorination of trichloroethylene and penta- and hexachloroethanes. The roles of Thr319, Glu318, and Lys250 in the catalysis with chlorinated hydrocarbons are discussed in association with reaction mechanisms.
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