title: Measuring dimethylallyl diphosphate available for isoprene synthesis.
abstract: Dimethylallyl diphosphate (DMADP) is a central metabolite in isoprenoid metabolism, but it is difficult to measure. Three different methods for measuring DMADP are compared, and a new method based on the conversion of DMADP to isoprene using recombinant isoprene synthase is introduced. Mass spectrometry is reliable but does not distinguish between DMADP and isopentenyl diphosphate. Acid hydrolysis is reliable for measuring DMADP in bacterial extracts but overestimates DMADP in plant samples. To measure the DMADP in chloroplasts, light minus dark measurements are normally used. Chloroplast DMADP amounts measured using acid hydrolysis and a mass spectrometric method were comparable in this assay. Post-illumination isoprene emission tended to slightly overestimate chloroplast DMADP concentration. The DMADP pool size in bacteria is highly regulated, consistent with previous observations made with plants. DMADP is a very labile metabolite, but four methods described here allow measurements of samples from plants and bacteria. The use of recombinant isoprene synthase can greatly simplify the analysis. The various techniques tested here have advantages and disadvantages, and it is useful to have more than one method available when studying biological isoprene production.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Sean E
authors.initials: SE
authors.lastname: Weise
authors.forename: Ziru
authors.initials: Z
authors.lastname: Li
authors.forename: Allison E
authors.initials: AE
authors.lastname: Sutter
authors.forename: Alex
authors.initials: A
authors.lastname: Corrion
authors.forename: Aparajita
authors.initials: A
authors.lastname: Banerjee
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Sharkey
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D002070:Butadienes / Q000378:metabolism*; D002736:Chloroplasts / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005052:Eucalyptus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D045782:Hemiterpenes / Q000032:analysis* / Q000378:metabolism*; D006868:Hydrolysis; D009943:Organophosphorus Compounds / Q000032:analysis* / Q000378:metabolism; D010420:Pentanes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D002070:Butadienes; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D010420:Pentanes; D011994:Recombinant Proteins; C005059:isoprene; C043060:3,3-dimethylallyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C093854:isoprene synthase