Cytochrome P450 oxygenases of Taxol biosynthesis
Cytochrome P450 oxygenases of Taxol biosynthesis
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DOI：
10.1007/s11101-006-9006-4
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发表时间：
2006-01-01
影响因子：
7.7
通讯作者：
Croteau, Rodney
中科院分区：
文献类型：
--
作者：
Kaspera, Rudiger;Croteau, Rodney
细胞色素 P450 单加氧酶在二萜类抗癌药物紫杉醇的生物合成中发挥着重要作用，因为它们似乎构成了红豆杉 (Taxus) 物种中该途径 19 个酶促步骤的大约一半。经典的生化和分子方法的结合，包括无细胞酶研究和 mRNA 逆转录聚合酶链式反应 (RT-PCR) 的差异显示，以及对诱导的尖齿虎细胞 cDNA 文库进行同源搜索和随机测序，发现了六种新型细胞色素 P450 紫杉烷（紫杉烷二萜）羟化酶。这些基因彼此之间表现出异常高的序列相似性 (> 70%)，但与其他植物 P450 的相似性较低 (< 30%)，并且与其他植物 P450 具有显着的进化距离。尽管它们高度相似，但对这些羟化酶的功能分析表明，它们具有独特的底物特异性，导致紫杉烷核心在 C5 处初始羟基化后生物合成途径发生早期分叉，从而形成竞争但相互关联的分支的生物合成网络。在无法获得预测的紫杉烷前体的情况下，使用替代底物导致发现了两种核心加氧酶：2α-羟化酶和7β-羟化酶。这种通用方法可以加速对现有 P450 基因家族候选 cDNA 的功能分析，以确定该复杂途径的剩余氧合步骤。
Cytochrome P450 monooxygenases play a prominent role in the biosynthesis of the diterpenoid anticancer drug Taxol, as they appear to constitute about half of the 19 enzymatic steps of the pathway in yew (Taxus) species. A combination of classical biochemical and molecular methods, including cell-free enzyme studies and differential-display of mRNA-reverse transcription polymerase chain reaction (RT-PCR) combined with a homology-based searching and random sequencing of a cDNA library from induced T. cuspidata cells, led to the discovery of six novel cytochrome P450 taxoid (taxane diterpenoid) hydroxylases. These genes show unusually high sequence similarity with each other (> 70%) but low similarity (< 30%) to, and significant evolutionary distance from, other plant P450s. Despite their high similarity, functional analysis of these hydroxylases demonstrated distinctive substrate specificities responsible for an early bifurcation in the biosynthetic pathway after the initial hydroxylation of the taxane core at C5, leading into a biosynthetic network of competing, but interconnected, branches. The use of surrogate substrates, in cases where the predicted taxoid precursors were not available, led to the discovery of two core oxygenases, the 2 alpha- and the 7 beta-hydroxylase. This general approach could accelerate the functional analysis of candidate cDNAs from the extant family of P450 genes to identify the remaining oxygenation steps of this complex pathway.