Cytochrome P450 taxadiene 5α-hydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis
Cytochrome P450 taxadiene 5α-hydroxylase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis
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DOI：
10.1016/j.chembiol.2004.02.022
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发表时间：
2004-03-01
影响因子：
--
通讯作者：
Croteau, R
中科院分区：
文献类型：
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作者：
Jennewein, S;Long, RM;Croteau, R
红豆杉属抗癌药物紫杉醇生物合成的第一步是细胞色素P450介导的二萜烯前体化合物Taxa-4(5)，11(12)-diene-Taxa-4(20)，11(12)-dien-5α-ol的羟基化(双键迁移)。利用诱导的红豆杉细胞文库，采用基于同源性的克隆策略，获得了编码紫杉烯5α-羟基酶的cDNA，并在酵母和昆虫细胞中进行了功能表达。重组酶能有效地利用紫杉醇-4(5)，11(12)-二烯和紫杉醇-4(20)，11(12)-二烯作为不定底物合成紫杉醇。该羟基酶在序列和性质上与紫杉醇生物合成的其他细胞色素P450加氧酶相似。这两种紫杉烯异构体在紫杉烯4(20)，11(12)-二烯-5α-醇的形成中的利用是新的，这表明了一种反应机理，包括通过选择性氧插入的混杂自由基提取而不是天然底物的C4，C5-烯烃的环氧化和生成的紫杉烯-11(12)-烯-4，5环氧化物的烯丙基重排。
The first oxygenation step in the biosynthesis of the anticancer drug taxol in Taxus species is the cytochrome P450-mediated hydroxylation (with double bond migration) of the diterpene olefin precursor taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5alpha-ol. A homology-based cloning strategy, employing an induced Taxus cell library, yielded a cDNA encoding taxadiene 5alpha-hydroxylase, which was functionally expressed in yeast and insect cells. The recombinant enzyme was characterized and shown to efficiently utilize both taxa-4(5),11(12)-diene and taxa-4(20),11(12)diene (as an adventitious substrate) to synthesize taxa4(20),11(12)-dien-5alpha-ol. This hydroxylase resembles, in sequence and properties, other cytochrome P450 oxygenases of taxol biosynthesis. The utilization of both taxadiene isomers in the formation of taxa4(20),11(12)-dien-5alpha-ol is novel, suggesting a reaction mechanism involving promiscuous radical abstraction with selective oxygen insertion rather than epoxidation of the C4,C5-alkene of the natural substrate and allylic rearrangement of the resulting taxa-11(12)-en-4,5epoxide.