Molecular cloning of a cytochrome P450 taxane 10β-hydroxylase cDNA from Taxus and functional expression in yeast
Molecular cloning of a cytochrome P450 taxane 10β-hydroxylase cDNA from Taxus and functional expression in yeast
复制标题
DOI：
10.1073/pnas.98.4.1501
复制
发表时间：
2001-02-13
影响因子：
11.1
通讯作者：
Croteau, RB
中科院分区：
文献类型：
--
作者：
Schoendorf, A;Rithner, CD;Croteau, RB
紫杉醇生物合成的早期步骤包括香叶基香叶基二磷酸环化为紫杉-4（5），11（12）-二烯，随后在C5处进行细胞色素P450介导的羟基化，该中间体的乙酰化，以及在C10处进行第二次细胞色素P450依赖性羟基化，产生紫杉二烯-5 α-乙酰氧基-10 β-醇.该途径的后续步骤涉及额外的细胞色素P450催化的氧化和CoA依赖性酰化。细胞色素P450加氧酶的反向遗传克隆的有限可行性导致使用诱导的紫杉醇生产的红豆杉细胞培养物和基于mRNA-逆转录-PCR的差异显示的方法的开发。其最终提供了13个独特但密切相关的细胞色素P450序列的全长形式。这些酶在酵母中的功能性表达通过原位分光光度法结合通过饲喂紫杉烷底物体内筛选加氧酶活性来监测。这种策略产生了紫杉烷代谢酶家族，并揭示了紫杉烷10 β-羟化酶为1494-bp cDNA，其编码498-残基的细胞色素P450，其能够将紫杉二烯乙酸酯转化为10 β-羟基衍生物;后一途径中间体的身份通过色谱和光谱方法确认。10 β-羟化酶代表紫杉醇生物合成的初始细胞色素P450基因，其通过提供途径的剩余加氧酶的途径分离。
The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5 alpha -acetoxy-10 beta -ol. Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations, The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences. Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates, This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10 beta -hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10 beta -hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means. The 10 beta -hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway.