Functional characterization of CYP1A9 and CYP1C1 from Anguillus japonica

Tomohide Uno; Chiho Izumi; Shinji Takenaka; Takeshi Yanase; Hiromasa Imaishi; Kengo Kanamaru; Hiroshi Yamagata; Yoshio Kaminishi; Takao Itakura

doi:10.1016/j.etap.2015.07.006

Functional characterization of CYP1A9 and CYP1C1 from Anguillus japonica

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

Authors

Tomohide Uno¹, Chiho Izumi², Shinji Takenaka³, Takeshi Yanase², Hiromasa Imaishi⁴, Kengo Kanamaru², Hiroshi Yamagata², Yoshio Kaminishi⁵, Takao Itakura⁵

Affiliations

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.

PMID: 26233561
DOI: 10.1016/j.etap.2015.07.006

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Anguilla / genetics*
Animals
Binding Sites
Coumarins / metabolism
Cytochrome P-450 Enzyme System / chemistry
Cytochrome P-450 Enzyme System / genetics*
Cytochrome P-450 Enzyme System / metabolism*
Escherichia coli / genetics
Escherichia coli / metabolism
Fish Proteins / chemistry
Fish Proteins / genetics*
Fish Proteins / metabolism*
Mutation
Phenacetin / metabolism
Progesterone / metabolism
Substrate Specificity

Substances

Coumarins
Fish Proteins
7-ethoxycoumarin
Progesterone
Cytochrome P-450 Enzyme System
Phenacetin