Publications

• Casbene synthetase: regulation of phytoalexin biosynthesis in Ricinus communis L. seedlings. Purification of casbene synthetase and regulation of its biosynthesis during elicitation.

  Moesta P., West C.A.

  Castor bean (Ricinus communis L.) seedlings responded to stress by producing the antifungal diterpene, casbene. Casbene synthetase, the enzyme catalyzing the production of casbene from geranylgeranyl pyrophosphate, was purified 4700-fold to a final specific activity of 4.2 nkat/mg protein by a com ... >> More

  Castor bean (Ricinus communis L.) seedlings responded to stress by producing the antifungal diterpene, casbene. Casbene synthetase, the enzyme catalyzing the production of casbene from geranylgeranyl pyrophosphate, was purified 4700-fold to a final specific activity of 4.2 nkat/mg protein by a combination of ion-exchange and dye-ligand chromatographic procedures. Approximately 500 micrograms of purified enzyme was recovered from 1600 seedlings that had been infected with the fungus, Rhizopus stolonifer. The purified enzyme showed a single protein band, by Ag staining, of Mr 59,000 +/-1000 after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoretic analysis of the immunoprecipitate obtained from a crude enzyme extract and polyclonal rabbit antibodies raised against the purified enzyme revealed no contaminants or cross-reacting components. In vitro translation of polysomal RNA pools obtained from healthy castor bean seedlings and seedlings at various times after exposure to pectic fragment elicitors coupled with immunoprecipitation showed that healthy seedlings have nondetectable levels of casbene synthetase mRNA and that seedlings exposed to elicitor show a rapid increase in casbene synthetase mRNA which reaches a maximum after 6 h. Casbene synthetase activity increases to a maximum 10 h after elicitation under comparable conditions. These results show that increases in the activity of mRNA for casbene synthetase after elicitation by pectic fragments precede the appearance of casbene synthetase activity as would be expected if the enzyme were being synthesized de novo. << Less

  Arch Biochem Biophys 238:325-333(1985) [PubMed] [EuropePMC]

• Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.

  Luo D., Callari R., Hamberger B., Wubshet S.G., Nielsen M.T., Andersen-Ranberg J., Hallstroem B.M., Cozzi F., Heider H., Lindberg Moeller B., Staerk D., Hamberger B.

  The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogen ... >> More

  The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogenase-mediated cyclization step in the biosynthetic pathway of Euphorbia factors, illustrating the origin of the intramolecular carbon-carbon bonds present in lathyrane and ingenane diterpenoids. This unconventional cyclization describes the ring closure of the macrocyclic diterpene casbene. Through transcriptomic analysis of E. lathyris L. mature seeds and in planta functional characterization, we identified three enzymes involved in the cyclization route from casbene to jolkinol C, a lathyrane diterpene. These enzymes include two cytochromes P450 from the CYP71 clan and an alcohol dehydrogenase (ADH). CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and 5-oxidation of casbene, respectively. When coupled with these P450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes dehydrogenation of the hydroxyl groups, leading to the subsequent rearrangement and cyclization. The discovery of this nonconventional cyclization may provide the key link to complete elucidation of the biosynthetic pathways of ingenol mebutate and other bioactive macrocyclic diterpenoids. << Less

  Proc. Natl. Acad. Sci. U.S.A. 113:E5082-E5089(2016) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.