Heterologous expression of CYP2K1 and identification of the expressed protein (BV-CYP2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase.
Heterologous expression of CYP2K1 and identification of the expressed protein (BV-CYP2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase.
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发表时间：
2000-11
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通讯作者：
Yea-Huey Yang;Cristobal L. Miranda;M. Henderson;J. Wang‐Buhler;Donald R. Buhler
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作者：
Yea-Huey Yang;Cristobal L. Miranda;M. Henderson;J. Wang‐Buhler;Donald R. Buhler
LMC2是在性未成熟虹鳟（Onchorynchus mykiss）中最丰富的组成性表达的肝细胞色素P450，也是激活致癌物黄曲霉毒素B1 （AFB1）的同工酶。该P450已被克隆，测序，并指定为CYP2K1。本报告描述了酶活性CYP2K1 （BV-CYP2K1）在杆状病毒（Sf9）昆虫细胞中的异源表达及其催化和免疫反应性表征，并与先前纯化的LMC2 P450进行了比较。在大鼠nadph -细胞色素P450还原酶存在的情况下，表达CYP2K1酶和LMC2的Sf9细胞均能催化月桂酸的羟基化和AFB1的环氧化。LMC2和BV-CYP2K1都催化月桂酸的氧化，主要在（omega-1）位点，少量在（omega-2）位点。当P450孵育混合物中含有AFB1、谷胱甘肽（GSH）和小鼠肝细胞质或纯化的大鼠谷胱甘肽转移酶时，通过AFB1环氧化物-谷胱甘肽偶联物的出现间接表明AFB1环氧化物的形成。当BV-CYP2K1和纯化的LMC2产生的AFB1环氧化物- gsh偶联物用手性色谱柱分析时，它的保留时间与CYP3A4产生的保留时间相同，CYP3A4是一种已知只形成AFB1外环氧化物的人类P450。因此，这些结果证实，cdna表达的CYP2K1蛋白在催化和免疫上与纯化的鳟鱼LMC2相同，这两种酶主要产生高度致癌的立体异构体外环氧化物形式的AFB1。
LMC2 is the most abundant constitutively expressed hepatic cytochrome P450 found in sexually immature rainbow trout (Onchorynchus mykiss) and is also the isozyme that activates the carcinogen aflatoxin B1 (AFB1). This P450 has been cloned, sequenced, and designated as CYP2K1. The present report describes the heterologous expression of enzymatically active CYP2K1 (BV-CYP2K1) in baculovirus Spodoptera frugiperda (Sf9) insect cells and its catalytic and immunoreactivity characterization in comparison with that of the previously purified LMC2 P450. Homogenates of Sf9 cells expressing the CYP2K1 enzyme and LMC2 both catalyzed the hydroxylation of lauric acid and the epoxidation of AFB1 in the presence of rat NADPH-cytochrome P450 reductase. Both LMC2 and BV-CYP2K1 catalyzed the oxidation of lauric acid primarily at the (omega-1) position plus small amounts at the (omega-2) position. Formation of AFB1 epoxide was shown indirectly by the appearance of an AFB1 epoxide-glutathione conjugate when P450 incubation mixtures contained AFB1, glutathione (GSH) together with mouse liver cytosol or purified rat GSH-transferase. When the AFB1 epoxide-GSH conjugate produced by BV-CYP2K1 and purified LMC2 was analyzed by HPLC using a chiral column, it had a retention time identical to that produced by CYP3A4, a human P450 known to form exclusively the AFB1 exo-epoxide. These results, therefore, confirm that the cDNA-expressed CYP2K1 protein is catalytically and immunologically identical to purified trout LMC2 and that these two enzymes produce primarily the highly carcinogenic stereoisomeric exo-epoxide form of AFB1.