title: Alkoxyresorufin (methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin) O-dealkylase activities by in vitro-expressed cytochrome P450 1A4 and 1A5 from common cormorant (Phalacrocorax carbo).
abstract: Here we report the inter-paralog comparison of cytochrome P4501A (CYP1A) catalytic function in common cormorant (Phalacrocorax carbo) using the recombinant proteins synthesized by yeast-based vector system. CYP1A4 and CYP1A5 proteins from common cormorant were heterologously expressed in yeast Saccaromyces cerevisiae. Kinetic analyses revealed that among alkoxyresorufin (methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin) O-dealkylase (AROD) activities V(max) value for ethoxyresorufin O-deethylase (EROD) activity was the highest for both enzymes, reaching 0.91+/-0.034 and 1.8+/-0.043 nmol/min/nmol CYP for CYP1A4 and CYP1A5, respectively. Similar results were obtained for the catalytic efficiencies represented as the ratios of V(max) to K(m) (V(max)/K(m)). Meanwhile, distinct substrate preferences were also observed; CYP1A4 had V(max) and V(max)/K(m) values for benzyloxyresorufin O-debenzylase (BROD) activity 12- and 46-fold greater than CYP1A5, respectively, while CYP1A5 was about 13- and 4.5-fold more efficient in methoxyresorufin O-demethylase (MROD) activity than CYP1A4. The K(m) values showed no significant change among MROD, EROD, pentoxyresorufin O-depenthylase (PROD) and BROD activities for both enzymes, except for significant differences between PROD and other three activities for CYP1A4. Comparing the results in the present study with previous studies addressing chicken and rat CYP1A enzymes, it is also clear that CYP1A orthologs have different catalytic preferences for AROD activities between cormorant and rat and even between cormorant and chicken. Variations in CYP1A catalytic function between cormorant CYP1A paralogs and between CYP1A orthologs from cormorant and other species indicate that enzymatic properties should be characterized on the basis not only of a limited model species such as chicken, but also of multiple species to further understand the mechanism underlying differences in substrate selectivity and the interaction with environmental contaminants in avian species.
authors.affiliation: Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kubota
authors.forename: Eun-Young
authors.initials: EY
authors.lastname: Kim
authors.forename: Hisato
authors.initials: H
authors.lastname: Iwata
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001717:Birds; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; C076307:methoxyresorufin-O-demethylase; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; C108015:cytochrome P-450 CYP1A5