Measuring the Activity of 1-Deoxy-D-Xylulose 5-Phosphate Synthase, the First Enzyme in the MEP Pathway, in Plant Extracts
The first enzyme in the methylerythritol phosphate (MEP) pathway is 1-deoxy-d-xylulose 5-phosphate (DXP) synthase (DXS). As such this enzyme is considered to be important in the control of plastidial isoprenoid production. Measuring the activity of DXS in plant extracts is therefore crucial to understanding the regulation of the MEP pathway. Due to the relatively low amounts of DXS, the activity of this enzyme can only be measured using highly sensitive analytical equipment. Here, a method is described to determine the DXS enzyme activity in a crude plant extract, by measuring DXP production directly using high performance liquid chromatography linked to a tandem triple quadrupole mass spectrometry detector (LC-MS/MS).
KeywordsDXS Enzyme assay Isoprenoid biosynthesis LC-MS/MS
We thank Bettina Raguschke for technical assistance and Felix Rohdich, Adelbert Bacher and Wolfgang Eisenreich for the kind gift of 13C labelled DXP. The project was funded by the Max Planck Gesellschaft.
- 3.Lois LM, Campos N, Putra SR et al (1998) Cloning and characterization of a gene from Escerichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of d-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamine, and pyridoxol biosynthesis. Proc Natl Acad Sci U S A 95:2105–2110PubMedCentralPubMedCrossRefGoogle Scholar