Publications

• Isoprene synthase activity parallels fluctuations of isoprene release during growth of Bacillus subtilis.

  Sivy T.L., Shirk M.C., Fall R.

  Isoprene is a volatile metabolite of uncertain function in plants, animals, and bacteria. Here, we demonstrate that the isoprene-producing bacterium, Bacillus subtilis, contains an isoprene synthase activity that catalyzes dimethylallyl diphosphate-dependent isoprene formation. Although the enzyme ... >> More

  Isoprene is a volatile metabolite of uncertain function in plants, animals, and bacteria. Here, we demonstrate that the isoprene-producing bacterium, Bacillus subtilis, contains an isoprene synthase activity that catalyzes dimethylallyl diphosphate-dependent isoprene formation. Although the enzyme was very labile, it was demonstrated in both permeabilized cells and in partially purified cell extracts. Its activity was optimal at pH 6.2, required low levels of a divalent cation, and appears distinct from chloroplast isoprene synthases. When grown in a bioreactor, B. subtilis cells released isoprene in three distinct phases; using permeabilized cells, it was shown that isoprene synthase activity rose and fell in parallel with each phase. These results suggest that isoprene synthesis is highly regulated in B. subtilis and further research in this model system may shed light on the role of isoprene formation in biological systems. << Less

  Biochem Biophys Res Commun 294:71-75(2002) [PubMed] [EuropePMC]

• First isolation of an isoprene synthase gene from poplar and successful expression of the gene in Escherichia coli.

  Miller B., Oschinski C., Zimmer W.

  For the first time, the complete functional gene for isoprene synthase has been isolated from poplar (Populus alba x Populus tremula). The gene was quite similar to known limonene and other monoterpene synthases, but was found to specifically catalyze the formation of isoprene from the precursor d ... >> More

  For the first time, the complete functional gene for isoprene synthase has been isolated from poplar (Populus alba x Populus tremula). The gene was quite similar to known limonene and other monoterpene synthases, but was found to specifically catalyze the formation of isoprene from the precursor dimethylallyl diphosphate with only a marginal activity for the formation of the monoterpene limonene from geranyl diphosphate as compared with limonene synthases. Omitting the part of the gene that putatively encoded the signal peptide necessary for transport into the chloroplast led to an enhanced rate of isoprene formation by the recombinant protein. << Less

  Planta 213:483-487(2001) [PubMed] [EuropePMC]

• Gene expression and characterization of isoprene synthase from Populus alba.

  Sasaki K., Ohara K., Yazaki K.

  Isoprene synthase cDNA from Populus alba (PaIspS) was isolated by RT-PCR. This PaIspS mRNA, which was predominantly observed in the leaves, was strongly induced by heat stress and continuous light irradiation, and was substantially decreased in the dark, suggesting that isoprene emission was regul ... >> More

  Isoprene synthase cDNA from Populus alba (PaIspS) was isolated by RT-PCR. This PaIspS mRNA, which was predominantly observed in the leaves, was strongly induced by heat stress and continuous light irradiation, and was substantially decreased in the dark, suggesting that isoprene emission was regulated at the transcriptional level. The subcellular localization of PaIspS protein with green fluorescent protein fusion was shown to be in plastids. PaIspS expressed in Escherichia coli was characterized enzymatically: it had an optimum pH of approximately 8.0, and an optimum temperature 40 degrees C. Its preference for divalent cations for its activity was also studied. << Less

  FEBS Lett. 579:2514-2518(2005) [PubMed] [EuropePMC]

• Biochemical properties of isoprene synthase in poplar (Populus x canescens).

  Schnitzler J.P., Zimmer I., Bachl A., Arend M., Fromm J., Fischbach R.J.

  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 lea ... >> More

  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. << Less

  Planta 222:777-786(2005) [PubMed] [EuropePMC]

• Structure of isoprene synthase illuminates the chemical mechanism of teragram atmospheric carbon emission.

  Koeksal M., Zimmer I., Schnitzler J.-P., Christianson D.W.

  The X-ray crystal structure of recombinant PcISPS (isoprene synthase from gray poplar hybrid Populus×canescens) has been determined at 2.7 Å resolution, and the structure of its complex with three Mg(2+) and the unreactive substrate analogue dimethylallyl-S-thiolodiphosphate has been determined at ... >> More

  The X-ray crystal structure of recombinant PcISPS (isoprene synthase from gray poplar hybrid Populus×canescens) has been determined at 2.7 Å resolution, and the structure of its complex with three Mg(2+) and the unreactive substrate analogue dimethylallyl-S-thiolodiphosphate has been determined at 2.8 Å resolution. Analysis of these structures suggests that the generation of isoprene from substrate dimethylallyl diphosphate occurs via a syn-periplanar elimination mechanism in which the diphosphate-leaving group serves as a general base. This chemical mechanism is responsible for the annual atmospheric emission of 100 Tg of isoprene by terrestrial plant life. Importantly, the PcISPS structure promises to guide future protein engineering studies, potentially leading to hydrocarbon fuels and products that do not rely on traditional petrochemical sources. << Less

  J. Mol. Biol. 402:363-373(2010) [PubMed] [EuropePMC]