title: Isopentenyl diphosphate and dimethylallyl diphosphate/isopentenyl diphosphate ratio measured with recombinant isopentenyl diphosphate isomerase and isoprene synthase.
abstract: Isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) are building units for all isoprenoids; thus, intracellular pool sizes of IDP and DMADP play important roles in living organisms. Several methods have been used to quantify the amount of DMADP or the combined amount of IDP plus DMADP, but measuring the DMADP/IDP ratio has been difficult. In this study, a method was developed to measure the ratio of DMADP/IDP. Catalyzed by a recombinant IDP isomerase (IDI) together with a recombinant isoprene synthase (IspS), IDP was converted to isoprene, which was then detected by chemiluminescence. With this method, the in vitro equilibrium ratio of DMADP/IDP was found to be 2.11:1. IDP and DMADP pools were significantly increased in Escherichia coli transformed with methylerythritol 4-phosphate pathway genes; the ratio of DMADP/IDP was 3.85. An E. coli strain transformed with IspS but no additional IDI had a lower DMADP level and a DMADP/IDP ratio of 1.05. Approximately 90% of the IDP and DMADP pools in light-adapted kudzu leaves were light dependent and so presumably were located in the chloroplasts; the DMADP/IDP ratios in chloroplasts and cytosol were the same as the in vitro ratio (2.04 in the light and 2.32 in the dark).
authors.affiliation: School of Life Science, Nanjing University, Nanjing 210093, China.
authors.forename: Changfang
authors.initials: C
authors.lastname: Zhou
authors.forename: Ziru
authors.initials: Z
authors.lastname: Li
authors.forename: Amy E
authors.initials: AE
authors.lastname: Wiberley-Bradford
authors.forename: Sean E
authors.initials: SE
authors.lastname: Weise
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Sharkey
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D019748:Carbon-Carbon Double Bond Isomerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D045782:Hemiterpenes / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology; D029908:Pueraria / Q000201:enzymology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C004809:isopentenyl pyrophosphate; C043060:3,3-dimethylallyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C093854:isoprene synthase; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase
keywords: Dimethylallyl diphosphate (DMADP); Isomerase equilibrium; Isopentenyl diphosphate (IDP); Isoprenoids; Recombinant enzyme