Krynetski E Y, Drutsa V L, Kovaleva I E, Luzikov V N
Chemistry Faculty, Moscow State University, Russia.
Pharmacogenetics. 1995 Apr;5(2):103-9. doi: 10.1097/00008571-199504000-00007.
In order to develop a model system for studying drug metabolism, we constructed recombinant yeast strains expressing human liver cytochromes P450. A high yield of cDNA-derived CYP2D6 was obtained, due to optimization of the initiation ATG codon context. The PCR-based site-mutagenesis method was used to introduce an AAA sequence immediately before the initiation codon resulting in increased translation of the GAL10-CYC1-derived mRNA. The use of a peptidase-deficient yeast strain also helped to increase the CYP2D6 content. A P450 content of 250 +/- 30 pmol per mg of microsomal protein was achieved. HPLC analysis confirmed that heterologously expressed CYP2D6 catalysed the oxidation of debrisoquine and dextromethorphan, two prototype substrates for CYP2D6. The Km for debrisoquine 4-hydroxylase was found to be 50 microM and Vmax 7.5 pmol mg-1 min-1. Dextromethorphan O-demethylase activity in CYP2D6-containing microsomes was characterized by Km 8.5 microM and Vmax 700 pmol mg-1 min-1. Biotransformation of debrisoquine and dextromethorphan was not detected in control microsomes. Yeast synthesizing CYP2D6 represents a useful in vitro system for studying xenobiotic metabolism.
为了开发一个用于研究药物代谢的模型系统，我们构建了表达人肝细胞色素P450的重组酵母菌株。由于起始ATG密码子上下文的优化，获得了高产的cDNA衍生的CYP2D6。基于PCR的定点诱变方法用于在起始密码子之前立即引入AAA序列，从而增加了GAL10-CYC1衍生mRNA的翻译。使用肽酶缺陷型酵母菌株也有助于增加CYP2D6的含量。每毫克微粒体蛋白的P450含量达到250±30 pmol。HPLC分析证实，异源表达的CYP2D6催化了异喹胍和右美沙芬的氧化，这两种是CYP2D6的原型底物。发现异喹胍4-羟化酶的Km为50μM，Vmax为7.5 pmol mg-1 min-1。含CYP2D6的微粒体中的右美沙芬O-脱甲基酶活性的特征为Km 8.5μM，Vmax 700 pmol mg-1 min-1。在对照微粒体中未检测到异喹胍和右美沙芬的生物转化。合成CYP2D6的酵母代表了一种用于研究异源物质代谢的有用体外系统。
