Generation of 2,3,7,8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis.
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.
Shinkyo
Raku
R
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto, Japan.
Sakaki
Toshiyuki
T
Takita
Teisuke
T
Ohta
Miho
M
Inouye
Kuniyo
K
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
D004147:Dioxins; D004785:Environmental Pollutants; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D019363:Cytochrome P-450 CYP1A1