CYP725A4 from yew catalyzes complex structural rearrangement of taxa-4(5),11(12)-diene into the cyclic ether 5(12)-oxa-3(11)-cyclotaxane
CYP725A4 from yew catalyzes complex structural rearrangement of taxa-4(5),11(12)-diene into the cyclic ether 5(12)-oxa-3(11)-cyclotaxane
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DOI：
10.1074/jbc.m708950200
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发表时间：
2008-03-07
影响因子：
4.8
通讯作者：
Tissier, Alain
中科院分区：
文献类型：
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作者：
Rontein, Denis;Onillon, Sandrine;Tissier, Alain
紫杉-4（5），11（12）-二烯是功能化紫杉烷如紫杉醇（一种成功的抗癌药物）的第一个确定的前体。紫杉烷的生物合成涉及几个氧化，其中一些已被证明是由细胞色素P-450加氧酶催化。紫杉-4（5），11（12）-二烯的C-5 α的羟基化被认为是这些氧化中的第一个，并且最近描述了编码紫杉-4（5），11（12）-二烯5 α-羟化酶（CYP 725 A4）的基因（Jennewein，S.，朗河M.，威廉姆斯河M.，和Croteau，R.等人（2004）Chem.Biol.11，379-387）。为了通过代谢工程方法生产紫杉醇途径的早期组分，将编码taxa-4（5），11（12）-diene synthase和CYP 725 A4的cDNA导入欧洲烟草（Nicotiana sylvestris）中，使其在毛状体细胞中特异性表达。它们的共表达没有导致产生预期的5 α-羟基紫杉-4（20），11（12）-二烯。相反，紫杉-4（5），11（12）-二烯定量转化为一种新的紫杉烷，纯化和表征。其结构经核磁共振分析确定为5（12）-氧杂-3（11）-环紫杉烷（OCT），其中紫杉-4（5），11（12）-二烯的8碳B环被分成两个稠合的5碳环。此外，OCT含有从分子的相对侧连接C-5和C-12的醚桥。OCT也是紫杉-4（5），11（12）-二烯与NADPH和从表达CYP 725 A4的重组酵母制备的微粒体孵育后获得的唯一主要产物。因此，紫杉-4（5），11（12）-二烯环系统的重排仅由CYP 725 A4介导，而不依赖于植物中存在的其他酶或因子。OCT的复杂结构使我们提出了一种涉及P-450催化中迄今未知的一系列事件的反应机制。
Taxa-4(5),11(12)-diene is the first committed precursor of functionalized taxanes such as paclitaxel, a successful anticancer drug. Biosynthesis of taxanes in yew involves several oxidations, a number of which have been shown to be catalyzed by cytochrome P-450 oxygenases. Hydroxylation of the C-5 alpha of taxa-4(5),11(12)-diene is believed to be the first of these oxidations, and a gene encoding a taxa-4(5),11(12)-diene 5 alpha-hydroxylase (CYP725A4) was recently described (Jennewein, S., Long, R. M., Williams, R. M., and Croteau, R. (2004) Chem. Biol. 11, 379-387). In an attempt to produce the early components of the paclitaxel pathway by a metabolic engineering approach, cDNAs encoding taxa-4(5),11(12)-diene synthase and CYP725A4 were introduced in Nicotiana sylvestris for specific expression in trichome cells. Their co-expression did not lead to the production of the expected 5 alpha-hydroxytaxa-4(20),11(12)-diene. Instead, taxa-4(5),11(12)-diene was quantitatively converted to a novel taxane that was purified and characterized. Its structure was determined by NMR analysis and found to be that of 5(12)-oxa-3(11)-cyclotaxane (OCT) in which the eight-carbon B-ring from taxa-4(5), 11(12)-diene is divided into two fused five-carbon rings. In addition, OCT contains an ether bridge linking C-5 and C-12 from opposite sides of the molecule. OCT was also the sole major product obtained after incubation of taxa-4(5),11(12)-diene with NADPH and microsomes prepared from recombinant yeast expressing CYP725A4. The rearrangement of the taxa-4(5),11(12)-diene ring system is thus mediated by CYP725A4 only and does not rely on additional enzymes or factors present in the plant. The complex structure of OCT led us to propose a reaction mechanism involving a sequence of events so far unknown in P-450 catalysis.