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Measuring dimethylallyl diphosphate available for isoprene synthesis
Sean E Weise1, Ziru Li, Allison E Sutter
1Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Analytical Biochemistry
|December 25, 2012
Summary
Measuring dimethylallyl diphosphate (DMADP), a key isoprenoid metabolite, is challenging. This study introduces a new method using recombinant isoprene synthase, simplifying DMADP analysis in plants and bacteria.
Area of Science:
- Biochemistry
- Metabolomics
- Plant Science
Background:
- Dimethylallyl diphosphate (DMADP) is a crucial intermediate in isoprenoid biosynthesis.
- Accurate quantification of DMADP is essential for understanding metabolic regulation but remains challenging due to its labile nature.
Purpose of the Study:
- To compare existing methods for DMADP measurement.
- To introduce and validate a novel method for DMADP quantification using recombinant isoprene synthase.
Main Methods:
- Comparison of mass spectrometry and acid hydrolysis for DMADP determination.
- Development of a new assay utilizing recombinant isoprene synthase to convert DMADP to isoprene.
- Analysis of DMADP in bacterial extracts and plant chloroplasts.
Main Results:
- Mass spectrometry cannot differentiate DMADP from its isomer, isopentenyl diphosphate.
- Acid hydrolysis overestimates DMADP in plant samples but is reliable for bacterial extracts.
- The novel isoprene synthase method offers a simplified approach for DMADP analysis, showing comparable results to existing methods in chloroplast assays.
- DMADP pool size regulation is observed in both bacteria and plants.
Conclusions:
- Multiple methods are necessary for accurate DMADP measurement across different biological systems.
- Recombinant isoprene synthase provides a valuable tool for simplifying DMADP quantification.
- Understanding DMADP dynamics is critical for studying biological isoprene production.
