Nonradioactive assay for detecting isoprenyl diphosphate synthase activity in crude plant extracts using liquid chromatography coupled with tandem mass spectrometry

作者：Nagel Raimund; Gershenzon Jonathan^*; Schmidt Axel

来源：Analytical Biochemistry, 2012, 422(1): 33-38.

DOI：10.1016/j.ab.2011.12.037

• Isoprenyl diphosphate synthases
• Prenyltransferases
• Terpenes
• LC-MS/MS
• Geranyl diphosphate
• Farnesyl diphosphate
• Geranylgeranyl diphosphate

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 now 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.

• 出版日期2012-3-1