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

二萜 红豆杉 生物合成 生物化学 ATP合酶 异源表达 立体化学 生物 化学 基因 重组DNA 植物
作者
D. Clive Williams,Mark R. Wildung,Alan Qingwu Jin,Dolan Dalal,John S. Oliver,Robert M. Coates,Rodney Croteau
出处
期刊:Archives of Biochemistry and Biophysics [Elsevier BV]
卷期号:379 (1): 137-146 被引量:105
标识
DOI:10.1006/abbi.2000.1865
摘要

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 (∼5%), and a product tentatively identified as verticillene (∼1%). Isotopically sensitive branching experiments utilizing (4R)-[4-2H1]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.
最长约 10秒,即可获得该文献文件

科研通智能强力驱动
Strongly Powered by AbleSci AI
更新
PDF的下载单位、IP信息已删除 (2025-6-4)

科研通是完全免费的文献互助平台,具备全网最快的应助速度,最高的求助完成率。 对每一个文献求助,科研通都将尽心尽力,给求助人一个满意的交代。
实时播报
张宝完成签到,获得积分10
1秒前
薄雪草完成签到,获得积分10
4秒前
大橙子发布了新的文献求助10
4秒前
快乐学习每一天完成签到 ,获得积分10
4秒前
薄荷味完成签到 ,获得积分0
6秒前
科研通AI2S应助笑林采纳,获得10
6秒前
无心的雅旋完成签到,获得积分10
7秒前
量子星尘发布了新的文献求助10
8秒前
Horizon完成签到 ,获得积分10
12秒前
Oliver完成签到 ,获得积分10
14秒前
Superman完成签到 ,获得积分10
17秒前
Tina酱完成签到 ,获得积分10
17秒前
琪琪完成签到,获得积分10
17秒前
双碳小王子完成签到,获得积分10
19秒前
smottom应助科研通管家采纳,获得10
21秒前
21秒前
明时完成签到,获得积分10
22秒前
杨瑞东完成签到 ,获得积分10
25秒前
yyyy完成签到,获得积分10
33秒前
缥缈的平卉完成签到 ,获得积分10
34秒前
量子星尘发布了新的文献求助10
45秒前
李爱国应助大橙子采纳,获得10
46秒前
magictoo发布了新的文献求助30
52秒前
54秒前
yang完成签到,获得积分10
54秒前
Minicoper发布了新的文献求助10
55秒前
快乐丸子完成签到,获得积分10
56秒前
简单而复杂完成签到,获得积分10
56秒前
大橙子发布了新的文献求助10
1分钟前
张牧之完成签到 ,获得积分10
1分钟前
冷冷暴力完成签到,获得积分10
1分钟前
YYY完成签到,获得积分10
1分钟前
1分钟前
gujian完成签到 ,获得积分10
1分钟前
帅气的秘密完成签到 ,获得积分10
1分钟前
自然函发布了新的文献求助10
1分钟前
冰冰双双完成签到,获得积分10
1分钟前
开心夏旋完成签到 ,获得积分0
1分钟前
我要读博士完成签到 ,获得积分10
1分钟前
活泼的大船完成签到,获得积分10
1分钟前
高分求助中
【提示信息,请勿应助】关于scihub 10000
Les Mantodea de Guyane: Insecta, Polyneoptera [The Mantids of French Guiana] 3000
徐淮辽南地区新元古代叠层石及生物地层 3000
The Mother of All Tableaux: Order, Equivalence, and Geometry in the Large-scale Structure of Optimality Theory 3000
Handbook of Industrial Diamonds.Vol2 1100
Global Eyelash Assessment scale (GEA) 1000
Picture Books with Same-sex Parented Families: Unintentional Censorship 550
热门求助领域 (近24小时)
化学 材料科学 医学 生物 工程类 有机化学 生物化学 物理 内科学 纳米技术 计算机科学 化学工程 复合材料 遗传学 基因 物理化学 催化作用 冶金 细胞生物学 免疫学
热门帖子
关注 科研通微信公众号,转发送积分 4038128
求助须知:如何正确求助?哪些是违规求助? 3575831
关于积分的说明 11373827
捐赠科研通 3305610
什么是DOI,文献DOI怎么找? 1819255
邀请新用户注册赠送积分活动 892655
科研通“疑难数据库(出版商)”最低求助积分说明 815022