Catalytic function of avian cytochrome P450 1A4 and 1A5 (CYP1A4 and CYP1A5) enzymes heterologously expressed using in vitro yeast system.
The present study clarifies the enzymatic properties of two avian cytochrome P4501A (CYP1A) paralogs, CYP1A4 and 1A5, using a yeast-based vector system. Recombinant CYP1A4 and 1A5 proteins from common cormorant (Phalacrocorax carbo) were expressed in yeast cells, and showed typical reduced CO-difference spectra with a peak at 446 nm. Kinetic analysis of O-dealkylase of methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin catalyzed by the CYP1A enzymes revealed that Vmax value for ethoxyresorufin-O-deethylase (EROD) activity was much higher than that for the other three O-dealkylase activities for both isozymes. Interestingly, remarkable substrate specificity of the CYP1As was observed for O-dealkylation of benzyloxyresorufin and methoxyresorufin; CYP1A4 was highly specific for catalyzing benzyloxyresorufin-O-debenzylase activity, whereas CYP1A5 was more efficient in catalyzing methoxyresorufin-O-demethylase activity. The present study also measured CYP1A-dependent EROD activity in the presence of 2,3,7,8-tetrachlorodibenzofuran (TCDF) to evaluate the ability of this dioxin-like congener to inhibit the EROD activity. One hundred nanomolar TCDF noncompetitively inhibited CYP1A5-dependent EROD activity, although no inhibitory effect was detected for CYP1A4-dependent EROD activity. These results indicate that the avian CYP1A paralogs have different affinities for substrate and inhibitor, thus suggesting their distinct physiological and toxicological roles.
Kubota
Akira
A
Division of Ecotoxicology, Center for Marine Environmental Studies, Ehime University, 2-5 Bunkyo-cho, Matsuyama, Japan.
Iwata
Hisato
H
Kim
Eun-Young
EY
D000818:Animals; D030161:Avian Proteins / Q000235:genetics* / Q000378:metabolism*; D001572:Benzofurans / Q000494:pharmacology; D001717:Birds / Q000235:genetics*; D019363:Cytochrome P-450 CYP1A1 / Q000187:drug effects / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D014874:Water Pollutants, Chemical / Q000494:pharmacology; D015003:Yeasts / Q000235:genetics*
D030161:Avian Proteins; D001572:Benzofurans; D014874:Water Pollutants, Chemical; D003577:Cytochrome P-450 Enzyme System; D019363:Cytochrome P-450 CYP1A1; C014211:2,3,7,8-tetrachlorodibenzofuran