Wildung M R, Croteau R
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
J Biol Chem. 1996 Apr 19;271(16):9201-4. doi: 10.1074/jbc.271.16.9201.
The committed step of taxol (paclitaxel) biosynthesis is catalyzed by taxa-4(5),11(12)-diene synthase, a diterpene cyclase responsible for transforming the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to the parent olefin with a taxane skeleton. To obtain the corresponding cDNA clone, a set of degenerate primers was constructed based on consensus sequences of related monoterpene, sesquiterpene, and diterpene cyclases. Two of these primers amplified a 83-base pair fragment that was cyclase-like in sequence and that was employed as a hybridization probe to screen a cDNA library constructed from poly(A)+ RNA extracted from Pacific yew (Taxus brevifolia) stems. Twelve independent clones with insert size in excess of 2 kilobase pairs were isolated and partially sequenced. One of these cDNA isolates was functionally expressed in Escherichia coli, yielding a protein that was catalytically active in converting geranylgeranyl diphosphate to a diterpene olefin that was confirmed to be taxa-4(5),11(12)-diene by combined capillary gas chromatography-mass spectrometry. The sequence specifies an open reading frame of 2586 nucleotides, and the complete deduced polypeptide, including a long presumptive plastidial targeting peptide, contains 862 amino acid residues and has a molecular weight of 98,303, compared with about 79,000 previously determined for the mature native enzyme. Sequence comparisons with monoterpene, sesquiterpene, and diterpene cyclases of plant origin indicate a significant degree of similarity between these enzymes; the taxadiene synthase most closely resembles (46% identity, 67% similarity) abietadiene synthase, a diterpene cyclase from grand fir.
紫杉醇生物合成的关键步骤由紫杉-4(5),11(12)-二烯合酶催化，这是一种二萜环化酶，负责将普遍存在的类异戊二烯中间体香叶基香叶基二磷酸转化为具有紫杉烷骨架的母体烯烃。为了获得相应的cDNA克隆，基于相关单萜、倍半萜和二萜环化酶的共有序列构建了一组简并引物。其中两个引物扩增出一个83碱基对的片段，其序列类似环化酶，并用作杂交探针来筛选由从太平洋紫杉（短叶红豆杉）茎中提取的聚腺苷酸加尾RNA构建的cDNA文库。分离出12个插入片段大小超过2千碱基对的独立克隆，并进行了部分测序。其中一个cDNA分离物在大肠杆菌中进行了功能表达，产生了一种蛋白质，该蛋白质在将香叶基香叶基二磷酸转化为二萜烯烃方面具有催化活性，通过毛细管气相色谱-质谱联用技术证实该二萜烯烃为紫杉-4(5),11(12)-二烯。该序列指定了一个2586个核苷酸的开放阅读框，完整推导的多肽包括一个长的推测性质体靶向肽，含有862个氨基酸残基，分子量为98,303，而之前测定的成熟天然酶分子量约为79,000。与植物来源的单萜、倍半萜和二萜环化酶的序列比较表明，这些酶之间存在显著程度的相似性；紫杉二烯合酶与冷杉中的二萜环化酶枞酸二烯合酶最为相似（46%的同一性，67%的相似性）。
