title: Characterization of chicken cytochrome P450 1A4 and 1A5: inter-paralog comparisons of substrate preference and inhibitor selectivity.
abstract: The chicken (Gallus gallus) is one of the most economically important domestic animals and also an avian model species. Chickens have two CYP1A genes (CYP1A4 and CYP1A5) which are orthologous to mammalian CYP1A1 and CYP1A2. Although the importance of chicken CYP1As in metabolism of endogenous compounds and xenobiotics is well recognized, their enzymatic properties, substrate preference and inhibitor selectivity remain poorly understood. In this study, functional enzymes of chicken CYP1A4 and CYP1A5 were successfully produced in Escherichia coli (E. coli). The substrate preference and inhibitor specificity of the two chicken CYP1As were compared. Kinetic results showed that the enzymatic parameters (K(m), V(max), V(max)/K(m)) for ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-debenzylase (BROD) differed between CYP1A4 and CYP1A5, while no significant difference was observed for methoxyresorufin O-demethylase (MROD). Lower K(m) of CYP1A4 for BROD suggests that CYP1A4 has a greater binding affinity to benzyloxyresorufin than either ethoxyresorufin or methoxyresorufin. The highest V(max)/K(m) ratio was seen in BROD activity for CYP1A4 and in MROD for CYP1A5 respectively. These results indicate that substrate preference of chicken CYP1As is more notably distinguished by BROD activity and CYP1A5 prefers shorter alkoxyresorufins resembling its mammalian ortholog CYP1A2. Differential patterns of MROD inhibition were observed between CYP1As and among the five CYP inhibitors (α-naphthoflavone, furafylline, piperonyl butoxide, erythromycin and ketoconazole). α-Naphthoflavone was determined to be a potent MROD inhibitor of both CYP1A4 and CYP1A5. In contrast, no or only a trace inhibitory effect (<15%) was observed by erythromycin at a concentration of 500 μM. Stronger inhibition of MROD activity was found in CYP1A5 than CYP1A4 by relatively small molecules α-naphthoflavone, piperonyl butoxide and furafylline. AROD kinetics and inhibition profiles between chicken CYP1A4 and CYP1A5 demonstrate that the two paralogous members of the CYP1A subfamily have distinct enzymatic properties, reflecting differences in the active site geometry between CYP1A4 and CYP1A5. These findings suggest that CYP1A4 and CYP1A5 play partially overlapping but distinctly different physiological and toxicological roles in the chicken.
authors.affiliation: State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Jiannan
authors.initials: J
authors.lastname: Yang
authors.forename: Junfeng
authors.initials: J
authors.lastname: An
authors.forename: Mei
authors.initials: M
authors.lastname: Li
authors.forename: Xin
authors.initials: X
authors.lastname: Hou
authors.forename: Xinghui
authors.initials: X
authors.lastname: Qiu
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D030161:Avian Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020134:Catalytic Domain; D002645:Chickens / Q000378:metabolism*; D019363:Cytochrome P-450 CYP1A1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004789:Enzyme Activation; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000378:metabolism; D004917:Erythromycin / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D010882:Piperonyl Butoxide / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D013806:Theophylline / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D030161:Avian Proteins; D004791:Enzyme Inhibitors; D010078:Oxazines; D004917:Erythromycin; C057754:benzyloxyresorufin; D003577:Cytochrome P-450 Enzyme System; D013806:Theophylline; C050131:furafylline; C076307:methoxyresorufin-O-demethylase; D001189:Aryl Hydrocarbon Hydroxylases; C416342:CYP1A4 protein, Gallus gallus; D019363:Cytochrome P-450 CYP1A1; D019362:Cytochrome P-450 CYP2B1; C108015:cytochrome P-450 CYP1A5; D010882:Piperonyl Butoxide