Biochemical properties of isoprene synthase in poplar (Populus x canescens).
Isoprene synthase (ISPS) catalyzes the elimination of pyrophosphate from dimethylallyl diphosphate (DMADP) forming isoprene, a volatile hydrocarbon emitted from many plant species to the atmosphere. In the present work, immunological techniques were applied to study and localize ISPS in poplar leaves (Populus x canescens). Immunogold labeling using polyclonal antibodies generated against His-tagged recombinant ISPS protein detected ca. 44% of ISPS in the stroma of the chloroplasts and ca. 56% of gold particles attached to the stromal-facing side of the thylakoid membranes. ISPS isolated from leaves exhibited the same biochemical properties as the recombinant ISPS without the plastid-targeting peptide heterologous expressed in E. coli, whereas an additional C- or N-terminal His-tag changed the biochemical features of the recombinant enzyme with regard to temperature, pH, and substrate dependence. In comparison to the closely related class of monoterpene synthases from angiosperms and ISPS of oaks, the most striking feature of the poplar ISPS is a cooperative substrate dependence which is characteristic to enzymes with positive substrate activation. The detection of four immunoreactive bands in poplar leaf extracts with isoelectric points from 5.0 to 5.5 and a native molecular weight of ca. 51 kDa give reason for future studies on post-translational modifications of ISPS.
Schnitzler
J-P
JP
Forschungszentrum Karlsruhe GmbH, Institut für Meteorologie und Klimaforschung, Atmosphärische Umweltforschung (IMK-IFU), Kreuzeckbahnstr. 19, 82467, Garmisch-Partenkirchen, Germany. joerg-peter.schnitzler@imk.fzk.de
Zimmer
I
I
Bachl
A
A
Arend
M
M
Fromm
J
J
Fischbach
R J
RJ
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007120:Immunochemistry; D007700:Kinetics; D032107:Populus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D014018:Tissue Distribution
D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C093854:isoprene synthase