title: Generation of 2,3,7,8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis.
abstract: Polychlorinated dibenzo-p-dioxins (PCDDs) are known as g environmental contaminants on account of the extreme toxicity. Among these compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is regarded as the most toxic one. The extremely high toxicity of 2,3,7,8-TetraCDD is based on its high affinity for Ah receptor and nearly undetectable metabolism in mammalian body. Based on our previous studies, we assumed that enlarging the space of substrate-binding pocket of rat CYP1A1 might generate the catalytic activity toward 2,3,7,8-TetraCDD. Large-sized amino acid residues located at putative substrate-binding sites of rat CYP1A1 were substituted for alanine by site-directed mutagenesis. Among eight mutants examined, the mutant in the putative F-G loop, F240A, showed metabolic activity toward 2,3,7,8-TetraCDD. HPLC and GC-MS analyses strongly suggested that the metabolite was 8-hydroxy-2,3,7-TriCDD. Ah receptor assay revealed that the affinity of 8-hydroxy-2,3,7-TriCDD for Ah receptor was less than 0.01% of 2,3,7,8-TetraCDD, indicating that the F240A-dependent metabolism resulted in remarkable detoxification of 2,3,7,8-TetraCDD. The novel 2,3,7,8-TetraCDD-metabolizing enzyme could be applicable to bioremediation of contaminated soils with dioxin, elimination of dioxin from foods, and clinical treatment for people who accidentally take dioxin into their systems.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto, Japan.
authors.forename: Raku
authors.initials: R
authors.lastname: Shinkyo
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Sakaki
authors.forename: Teisuke
authors.initials: T
authors.lastname: Takita
authors.forename: Miho
authors.initials: M
authors.lastname: Ohta
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D004147:Dioxins / Q000378:metabolism; D004785:Environmental Pollutants / Q000378:metabolism*; D006900:Hydroxylation; D007700:Kinetics; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism*; D051381:Rats; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D004147:Dioxins; D004785:Environmental Pollutants; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D019363:Cytochrome P-450 CYP1A1