State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
Comp Biochem Physiol C Toxicol Pharmacol. 2013 May;157(4):337-43. doi: 10.1016/j.cbpc.2013.02.005. Epub 2013 Mar 6.
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.
鸡（Gallus gallus）是最具经济重要性的家养动物之一，也是一种禽类模式物种。鸡有两个 CYP1A 基因（CYP1A4 和 CYP1A5），与哺乳动物的 CYP1A1 和 CYP1A2 同源。尽管鸡 CYP1A 在代谢内源性化合物和外源性化合物方面的重要性已得到充分认识，但它们的酶学特性、底物偏好和抑制剂选择性仍知之甚少。本研究在大肠杆菌（Escherichia coli）中成功表达了鸡 CYP1A4 和 CYP1A5 的功能酶。比较了两种鸡 CYP1A 的底物偏好和抑制剂特异性。动力学结果表明，CYP1A4 和 CYP1A5 的乙氧基resorufin O-去乙基酶（EROD）和苯氧resorufin O-去苯乙基酶（BROD）的酶学参数（K(m)、V(max)、V(max)/K(m)）不同，而甲氧基resorufin O-脱甲基酶（MROD）则没有明显差异。CYP1A4 对 BROD 的较低 K(m)表明，CYP1A4 与苯氧 resorufin 的结合亲和力大于乙氧基 resorufin 或甲氧基 resorufin。CYP1A4 的 BROD 活性和 CYP1A5 的 MROD 活性的 V(max)/K(m)比值最高。这些结果表明，鸡 CYP1A 的底物偏好通过 BROD 活性更为显著地区分，而 CYP1A5 更喜欢类似于其哺乳动物同源物 CYP1A2 的短烷氧基 resorufin。在 CYP1A 之间以及在五种 CYP 抑制剂（α-萘黄酮、呋喃西林、胡椒基丁氧基、红霉素和酮康唑）之间观察到 MROD 抑制的差异模式。α-萘黄酮被确定为 CYP1A4 和 CYP1A5 的强效 MROD 抑制剂。相比之下，红霉素在 500 μM 浓度下仅观察到抑制作用（<15%）或几乎没有抑制作用。与 CYP1A5 相比，相对小分子 α-萘黄酮、胡椒基丁氧基和呋喃西林对 CYP1A5 的 MROD 活性抑制作用更强。鸡 CYP1A4 和 CYP1A5 的 AROD 动力学和抑制谱表明，CYP1A 亚家族的两个同源成员具有不同的酶学特性，反映了 CYP1A4 和 CYP1A5 之间活性位点几何形状的差异。这些发现表明，CYP1A4 和 CYP1A5 在鸡中发挥部分重叠但明显不同的生理和毒理作用。
