Metabolism of linear and angular furanocoumarins by Papilio polyxenes CYP6B1 co-expressed with NADPH cytochrome P450 reductase.
Metabolism of linear and angular furanocoumarins by Papilio polyxenes CYP6B1 co-expressed with NADPH cytochrome P450 reductase.
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DOI：
10.1016/s0965-1748(03)00100-0
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发表时间：
2003-09
影响因子：
3.8
通讯作者：
Z. Wen;Liping Pan;M. Berenbaum;M. Schuler
中科院分区：
文献类型：
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作者：
Z. Wen;Liping Pan;M. Berenbaum;M. Schuler
微粒体细胞色素P450单加氧酶（P450）的异源表达中的一个挑战是满足其对从NADPH P450还原酶转移的电子的强制性要求。我们已经建立了多形凤蝶CYP 6 B1和家蝇P450还原酶在杆状病毒感染的Sf 9细胞中的共表达参数，该参数允许这两种组分的有效表达并显著增强该昆虫P450底物的代谢周转。这些表达条件使我们能够重新检查CYP 6 B1对宿主植物中存在的线性和角呋喃香豆素类的转换能力，用于专业毛虫P. polyxenes。CYP 6 B1和P450还原酶在等效病毒浓度下的共表达[MOI（感染复数）比为1]导致线性呋喃香豆素类黄毒素和peptide的转换率，其比单独表达CYP 6 B1获得的转换率增加32-33倍。角呋喃香豆素当归素的转换率也显著增加至4.76 nmol/min/nmol P450，而仅表达CYP 6 B1时几乎检测不到。底物结合分析表明，所有这三种化合物引起典型的I型结合光谱，但具有不同的幅度和亲和力，这表明每个基板的有效性，在协调与血红素铁。这些底物产生的高自旋态的相对比例与CYP 6 B1代谢这些呋喃香豆素的顺序一致，即黄毒素>补骨脂素>当归素。CYP 6 B1的这些不同活性表明，它可能是凤蝶科蝴蝶与其蜜蜂宿主植物之间共同进化军备竞赛的古老参与者。由于其处理一系列呋喃香豆素结构的能力，CYP 6 B1可能有助于多聚原杆菌在线性含呋喃香豆素植物的早期定殖，以及随后在角状含呋喃香豆素植物的定殖。
One challenge in the heterologous expression of microsomal cytochrome P450 monooxygenases (P450s) is fulfilling their obligatory requirement for electrons transferred from NADPH P450 reductase. We have established co-expression parameters for Papilio polyxenes CYP6B1 and house fly P450 reductase in baculovirus-infected Sf9 cells that allow for efficient expression of both components and significantly enhance metabolic turnover of this insect P450’s substrates. These expression conditions have allowed us to reexamine the turnover capacities of CYP6B1 toward linear and angular furanocoumarins present in the host plants for the specialist caterpillar P. polyxenes. Coexpression of CYP6B1 and P450 reductase at equivalent viral concentrations [MOI (multiplicity of infection) ratio of 1] results in turnover rates for the linear furanocoumarins xanthotoxin and psoralen, which are increased 32–33 fold over the turnover rates obtained with CYP6B1 expressed alone. The turnover rate for the angular furanocoumarin angelicin is also significantly increased to 4.76 nmol/min/nmol P450 compared to its barely detectable level obtained with CYP6B1 expressed alone. Substrate binding analyses indicate that all three of these compounds elicit typical type I binding spectra but with varying magnitudes and affinities that are indicative of each substrate’s effectiveness at coordinating with the heme iron. The relative proportions of high spin state generated with these substrates are consistent with CYP6B1 metabolizing these furanocoumarins in the rank order xanthotoxin>psoralen>angelicin. These differential activities for CYP6B1 suggest that it may have been an ancient participant in the coevolutionary arms race between papilionid butterflies and their apiaceous host plants. Due to its ability to handle a range of furanocoumarin structures, CYP6B1 may have contributed to P. polyxenes’ early colonization of linear furanocoumarin-containing plants and to its subsequent colonization of angular furanocoumarin-containing plants.