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Environ Toxicol Pharmacol. 2015 Sep;40(2):360-8. doi: 10.1016/j.etap.2015.07.006. Epub 2015 Jul 14.

Functional characterization of CYP1A9 and CYP1C1 from Anguillus japonica.

Author information

1
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.
2
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
3
Environmental Microbiology, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
4
Functional Analysis of Environmental Genes, Research Center for Environmental, Genomics, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
5
Laboratory of Marine Biotechnology, Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima, 890-0056, Japan.

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.

KEYWORDS:

CYP; Fish; HPLC; Monooxygenase; P450; Site-directed mutagenesis; progesterone

PMID:
26233561
DOI:
10.1016/j.etap.2015.07.006
[Indexed for MEDLINE]

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