Cloning and functional characterisation of a cis-muuroladiene synthase from black peppermint (Mentha x piperita) and direct evidence for a chemotype unable to synthesise farnesene
Cloning and functional characterisation of a cis-muuroladiene synthase from black peppermint (Mentha x piperita) and direct evidence for a chemotype unable to synthesise farnesene
复制标题
DOI：
10.1016/j.phytochem.2005.06.012
复制
发表时间：
2006-08-01
期刊：
影响因子：
3.8
通讯作者：
Field, Linda M.
中科院分区：
文献类型：
--
作者：
Prosser, Ian M.;Adams, Racheal J.;Field, Linda M.
利用针对（E）-β-法呢烯（E β F）合酶基因序列设计的寡核苷酸引物，从黑薄荷（Mentha x piperita）植物中克隆了两个cDNA序列（MxpSS 1和MxpSS 2）。MxpSS 1编码的蛋白质与E β F合酶具有96%的总体氨基酸序列同一性。在大肠杆菌中产生的重组MxpSS 1，在去除N-末端硫氧还蛋白融合物后，具有1.91 +/- 0.1 μ M的FPP的Km和0-18 s（-1）的k（cat），并将法呢基二磷酸（FPP）转化为四种产物，主要的两种是顺式-muurola-3，5-二烯（45%）和顺式-muurola-4（14），5-二烯（43%）。这是第一个顺式muuroladene合酶，其特征在于。MxpSS 2编码的蛋白质只有两个氨基酸不同于E β F合酶。重组MxpSS 2蛋白对FPP无活性。通过结构建模显示，在位置531处的两个突变之一（MxpSS 2中的亮氨酸和E β F合酶中的丝氨酸）发生在J-K环中，J-K环是已知在反应机制中重要的倍半萜烯脱氢酶结构的元件。通过定点诱变将531位的丝氨酸重新引入MxpSS 2恢复了E β F合酶活性（K.对于FPP为0.98 +/-0.12 pM，k（cat）0-1 s（-1）），证明了该残基在酶活性中的关键作用。通过GC-MS对用于克隆的植物的萜烯谱进行分析，发现E β F不存在，证实这种特定的薄荷化学型由于观察到的突变而失去了E β F合酶活性。(c)2005爱思唯尔有限公司保留所有权利。
Using oligonucleotide primers designed to the known gene sequence of an (E)-beta-farnesene (E beta F) synthase, two cDNA sequences (MxpSS1 and MxpSS2) were cloned from a black peppermint (Mentha x piperita) plant. MxpSS1 encoded a protein with 96% overall amino acid sequence identity with the E beta F synthase. Recombinant MxpSS1 produced in Escherichia coli, after removal of an N-terminal thioredoxin fusion, had a K-m for FPP of 1.91 +/- 0.1 mu M and k(cat), of 0-18 s(-1) and converted farnesyl diphosphate (FPP) into four products, the major two being cis-muurola-3,5-diene (45%) and cis-muurola-4(14),5-diene (43%). This is the first cis-muuroladiene synthase, to be characterised. MxpSS2 encoded a protein with only two amino acids differing from E beta F synthase. Recombinant MxpSS2 protein showed no activity towards FPP. One of the two mutations, at position 531 (leucine in MxpSS2 and serine in E beta F synthase) was shown, by structural modelling to occur in the J-K loop, an element of the structure of sesquiterpene synthases known to be important in the reaction mechanism. Reintroduction of the serine at position 531 into MxpSS2 by site-directed mutagenesis restored E beta F synthase activity (K. for FPP 0.98 +/- 0.12 pM, k(cat) 0-1 s(-1)), demonstrating the crucial role of this residue in the enzyme activity. Analysis, by GC-MS, of the sesquiterpene profile of the plant used for the cloning, revealed that E beta F was not present, confirming that this particular mint chemotype had lost E beta F synthase activity due to the observed mutations. (c) 2005 Elsevier Ltd. All rights reserved.