title: Functional characterization of CYP1A9 and CYP1C1 from Anguillus japonica.
abstract: We evaluated the metabolism of several herbicides and progesterone by two P450 proteins (CYP1A9 and CYP1C1) from Japanese eel (Anguilla japonica). Expression vectors harboring CYP1A9 and CYP1C1 sequences were introduced into Escherichia coli. E. coli membrane fractions were incubated with each substrate, and the metabolites were analyzed. CYP1A9 and CYP1C1 deethylated 7-ethoxycoumarin and phenacetin, and demethylated chlorotoluron, diuron, and linuron. CYP1C1 specifically hydroxlyated progesterone at the 6β and 16α positions. Five amino acids of CYP1A9 related to substrate binding were selected for mutation analyses [CYP1A9(F128A), CYP1A9(F229A), CYP1A9(F263A), CYP1A9(V387A), and CYP1A9(I391A)]. Two variants, CYP1A9(F229A) and CYP1A9(F128A), changed the ratio of 16α hydroxyprogesterone to 6β hydroxyprogesterone. Among all the variants, CYP1A9(F263A) showed the highest activity towards substrates used. CYP1A9(V387A) and CYP1A9(I391A) showed higher activities than that of CYP1A9 toward progesterone. The substrate specificity of CYP1A9 may be altered by replacing an amino acid related to substrate binding.
authors.affiliation: Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan. Electronic address: unotom@kobe-u.ac.jp.
authors.forename: Tomohide
authors.initials: T
authors.lastname: Uno
authors.affiliation: Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
authors.forename: Chiho
authors.initials: C
authors.lastname: Izumi
authors.affiliation: Environmental Microbiology, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Takenaka
authors.affiliation: Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yanase
authors.affiliation: Functional Analysis of Environmental Genes, Research Center for Environmental, Genomics, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Imaishi
authors.affiliation: Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Kanamaru
authors.affiliation: Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamagata
authors.affiliation: Laboratory of Marine Biotechnology, Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima, 890-0056, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kaminishi
authors.affiliation: Laboratory of Marine Biotechnology, Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima, 890-0056, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Itakura
mesh_terms: D000811:Anguilla / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D010615:Phenacetin / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D029941:Fish Proteins; C017299:7-ethoxycoumarin; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D010615:Phenacetin
keywords: CYP; Fish; HPLC; Monooxygenase; P450; Site-directed mutagenesis; progesterone