Department of Biochemistry, Max Planck Institute for Chemical Ecology, Jena, Germany.
Anal Biochem. 2012 Mar 1;422(1):33-8. doi: 10.1016/j.ab.2011.12.037. Epub 2012 Jan 3.
Terpenoids form the largest class of plant metabolites involved in primary and secondary metabolism. Isoprenyl diphosphate synthases (IDSs) catalyze the condensation of the C(5) terpenoid building blocks, isopentenyl diphosphate and dimethylallyl diphosphate, to form geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)). These branch point reactions control the flow of metabolites that act as precursors to each of the major terpene classes-monoterpenes, sequiterpenes, and diterpenes, respectively. Thus accurate and easily performed assays of IDS enzyme activity are critical to increase our knowledge about the regulation of terpene biosynthesis. Here we describe a new and sensitive nonradioactive method for carrying out IDS assays using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to detect the short-chain prenyl diphosphate products directly without dephosphorylation. Furthermore, we were able to separate cisoid and transoid isomers of both C(10) enzyme products (geranyl diphosphate and neryl diphosphate) and three C(15) products [(E,E)-, (Z,E)-, and (Z,Z)-farnesyl diphosphate]. By applying the method to crude protein extracts from various organs of Arabidopsis thaliana, Nicotiana attenuata, Populus trichocarpa, and Picea abies, we could determine their IDS activity in a reproducible fashion.
萜类化合物形成了参与初级和次级代谢的最大类植物代谢物。异戊烯二磷酸合酶（IDS）催化 C（5）萜类化合物构建块，异戊烯二磷酸和二甲基烯丙基二磷酸的缩合，形成香叶基二磷酸（C（10））、法呢基二磷酸（C（15））和香叶基香叶基二磷酸（C（20））。这些分支点反应控制着代谢物的流动，这些代谢物作为每个主要萜烯类别的前体，分别为单萜、倍半萜和二萜。因此，准确且易于进行的 IDS 酶活性测定对于增加我们对萜烯生物合成调控的认识至关重要。在这里，我们描述了一种新的灵敏的非放射性方法，用于使用液相色谱-串联质谱（LC-MS/MS）进行 IDS 测定，以直接检测短链 prenyl diphosphate 产物，而无需去磷酸化。此外，我们能够分离出 C（10）酶产物（香叶基二磷酸和橙花基二磷酸）和顺式和反式异构体以及三种 C（15）产物[（E，E）-、（Z，E）-和（Z，Z）-法呢基二磷酸]。通过将该方法应用于拟南芥、黄花烟草、白杨和黑云杉各种器官的粗蛋白提取物中，我们能够以可重复的方式确定它们的 IDS 活性。
