Heterologous expression and characterization of a "Pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction.
Heterologous expression and characterization of a "Pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction.
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DOI：
10.1006/abbi.2000.1865
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发表时间：
2000-07
影响因子：
3.9
通讯作者：
David C. Williams;M. Wildung;A. Q. Jin;Dolan Dalal;John S. Oliver;R. Coates;R. Croteau
中科院分区：
文献类型：
--
作者：
David C. Williams;M. Wildung;A. Q. Jin;Dolan Dalal;John S. Oliver;R. Coates;R. Croteau
来自红豆杉（Taxus）物种的二萜环化酶taxadiene合成酶将geranylgeranyl二磷酸转化为taxa-4(5),11(12)-二烯，这是抗癌药物紫杉醇生物合成的第一步。杉二烯合成酶被翻译为具有n端靶向序列的前蛋白，用于定位和在质体中加工。在大肠杆菌中，全长前蛋白的过表达和纯化受到宿主密码子使用、包涵体形成和与宿主伴侣的结合的影响，并且前蛋白的催化功能受损。由于无法直接确定转运肽成熟酶的切割位点，因此通过在合适的载体上表达相应的cdna，产生了一系列n端截断的酶，并对每种酶进行了纯化和动力学评价。删除多达79个残基得到功能蛋白；然而，删除93个或更多的氨基酸导致活性完全消除，这意味着氨基末端具有结构或催化作用。假成熟形式的杉二烯合成酶从前蛋白中删除了60个氨基酸，在表达水平、易于纯化、溶解度、稳定性和催化活性方面优于天然酶，其动力学与天然酶相当。除了主要产物taxa-4(5),11(12)-diene（94%）外，该酶还产生少量的异构体taxa-4(20), 11(12)-diene（约5%）和一种暂定为verticillene的产物（约1%）。利用(4R)-[4-(2)H(1)]geranylgeranyl二磷酸进行的同位素敏感分支实验证实，这两种杉二烯异构体和第三种(taxa-3（4),11(12)-二烯）是由相同的中间杉烯基c4 -碳正离子衍生而来。这些结果，以及酶利用2,7 -环香叶基二磷酸作为替代底物的失败，表明反应是通过二磷酸酯的初始电离和大环化到verticillyl中间体，然后是二次环化到紫杉基阳离子和去质子化（即，在B/ c环闭合之前形成a环）进行的。两种潜在的机制抑制剂用重组杉二烯合成酶进行了测试，但都没有提供时间依赖性失活，也没有提供超过适度的竞争性抑制。
The diterpene cyclase taxadiene synthase from yew (Taxus) species transforms geranylgeranyl diphosphate to taxa-4(5),11(12)-diene as the first committed step in the biosynthesis of the anti-cancer drug Taxol. Taxadiene synthase is translated as a preprotein bearing an N-terminal targeting sequence for localization to and processing in the plastids. Overexpression of the full-length preprotein in Escherichia coli and purification are compromised by host codon usage, inclusion body formation, and association with host chaperones, and the preprotein is catalytically impaired. Since the transit peptide-mature enzyme cleavage site could not be determined directly, a series of N-terminally truncated enzymes was created by expression of the corresponding cDNAs from a suitable vector, and each was purified and kinetically evaluated. Deletion of up to 79 residues yielded functional protein; however, deletion of 93 or more amino acids resulted in complete elimination of activity, implying a structural or catalytic role for the amino terminus. The pseudomature form of taxadiene synthase having 60 amino acids deleted from the preprotein was found to be superior with respect to level of expression, ease of purification, solubility, stability, and catalytic activity with kinetics comparable to the native enzyme. In addition to the major product, taxa-4(5),11(12)-diene (94%), this enzyme produces a small amount of the isomeric taxa-4(20), 11(12)-diene ( approximately 5%), and a product tentatively identified as verticillene ( approximately 1%). Isotopically sensitive branching experiments utilizing (4R)-[4-(2)H(1)]geranylgeranyl diphosphate confirmed that the two taxadiene isomers, and a third (taxa-3(4),11(12)-diene), are derived from the same intermediate taxenyl C4-carbocation. These results, along with the failure of the enzyme to utilize 2, 7-cyclogeranylgeranyl diphosphate as an alternate substrate, indicate that the reaction proceeds by initial ionization of the diphosphate ester and macrocyclization to the verticillyl intermediate, followed by a secondary cyclization to the taxenyl cation and deprotonation (i.e., formation of the A-ring prior to B/C-ring closure). Two potential mechanism-based inhibitors were tested with recombinant taxadiene synthase but neither provided time-dependent inactivation nor afforded more than modest competitive inhibition.