Bioconversion by functional P450 1A9 and P450 1C1 of Anguilla japonica.
We indicated that two P450s (1A9 and 1C1) from Japanese eel (Anguilla japonica) metabolized 7-ethoxycoumarin, 7-ethoxyresorufin, and flavanone. At first, we constructed expression vectors for two types of P450 (1A9 and 1C1). The reduced CO-difference spectra of Escherichia coli cells transformed with these plasmids showed Soret peaks (450 nm) that were typical of P450s. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolite(s) were extracted and analyzed by high-performance liquid chromatography and spectrofluorometer. Incubation of 50 nmol 7-ethoxyresorufin with P450 1C1 yielded 0.773 nmol of deethylated product, whereas 50 nmol 7-ethoxycoumarin resulted in 4.76 nmol. P450 1A9 metabolized 50 nmol of 7-ethoxyresorufin and 7-ethoxycoumarin to yield 6.54 and 20.9 nmol of deethylated product, respectively. Incubation of 50 nmol flavanone with P450 1C1 yielded 1.46 nmol and 0.69 nmol of products, whereas 50 nmol flavanone with P450 1A9 resulted in 1.10 nmol. In this system, 4'-hydroxy flavanones were formed by P450 1A9 and P450 1C1. P450 1A9 also metabolized 50 nmol of 17 beta-estradiol to yield 4.25 nmol of product. In this system, 2-hydroxy estradiol was formed by P450 1A9 using 17 beta-estradiol as a substrate. This study is the first to identify the substrates that P450 1C1 and 1A9 metabolize.
Uno
Tomohide
T
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe, Hyogo, 657-8501, Japan. unotom@kobe-u.ac.jp
Okamoto
Sota
S
Masuda
Satoko
S
Imaishi
Hiromasa
H
Nakamura
Masahiko
M
Kanamaru
Kengo
K
Yamagata
Hiroshi
H
El-Kady
Mohamed A H
MA
Kaminishi
Yoshio
Y
Itakura
Takao
T
D000811:Anguilla / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D003640:Dealkylation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D044950:Flavanones / Q000378:metabolism; D005822:Genetic Vectors; D006900:Hydroxylation; D007527:Isoenzymes / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D013379:Substrate Specificity; D014169:Transformation, Bacterial
D003374:Coumarins; D029941:Fish Proteins; D044950:Flavanones; D007527:Isoenzymes; D010078:Oxazines; C017299:7-ethoxycoumarin; D004958:Estradiol; C007649:ethoxyresorufin; D003577:Cytochrome P-450 Enzyme System; C028610:flavanone