Casbene synthetase from 67-hour seedings of Rkcihs communis L. which had been treated with Rhzp_5 stolonffer spores was prified 700-fold by a combination of ammonium sulfate fractionation, QAE A-50 Sephadex chromatography, and G-100 Sephadex chromatography. Polyacrylamide disc gel electrophoresis revealed that the purified fraction was heterogeneous. No casbene synthetase was detected in extracts of seedlings which had not been exposed to the fungal spores; maximum activity was obtained from seedlings 14 hours after exposure to spores.The pardally purified enzyme exbibited a broad pH optimum from pH 7.5 to 9.0 with half-maximal activty at pH 6.0 and 9.8. Chromatography on a calibrated Sephadex G-100 column nicated a molecular weight of 53,000 ± 3,000 for casbene synthetase. Concentrations of Mg2' above 5 mm gave maximal stimulation of the activity. Mn2+ was much less effective and was inhibitory at concentrations above 0.2 IM. The Km for geranylgeranyl pypsphate was estimated as 1.9 FM. The activity was inhibited 50% by 2.5 mm N-ethylmaleimlde; 10 mM iodoacetamide was not inhibitory. N,N-Dimethylaminoethyl-2,phenylpentanoate (SKF-525A) and the growth retardant 2'-Isopropyl-4'4trmethylammonum chloride)-5'-methylphenyl piperdi1ne--carboxylate (Amo-1618) were ineffective inhibitors of casbene synthetase, but the growth retardant tributyl-2,4-dichlorobenzylphosphonlum chloride (Phospbon D) at a concentration of 5 pM inhibited the activity by 55%.A cell-free system containing soluble enzymes from castor beans has been shown to synthesize five diterpene hydrocarbons from both mevalonic acid and geranylgeranyl pyrophosphate (17,18 system to produce casbene is greatly increased when the seedlings are exposed to cultures of Aspergillus niger or Rhizopus stolonifer (23) or to a partiafly purified elicitor isolated from culture filtrates of R stolonifer (24). In addition, casbene at a concentration of 10 ,ug/ml is inhibitory toward the growth of A. niger (23). It seems that casbene possesses some of the major characteristics of a phytoalexin as originally defined (6,13). None among the other known phytoalexins are hydrocarbons, and the momilactones are the only other diterpenoid substances proposed as phytoalexins (4).Most of the pathways postulated for the biosynthesis of known phytoalexins are complex with many steps involved. However, casbene is a product of a single enzyme-catalyzed transformation of GGPP, which serves as a branchpoint metabolite for several biosynthetic pathways in the castor bean. Thus, the castor bean system is ideal for the study of the enzymology and regulatory features of the production of a stress metabolite. Very little work has been reported on the enzymology associated with phytoalexin production, and none on an enzyme whose product is the phytoalexin itself. Uritani's group (15,25,26) has partially purified and measured changes in activities of some of the enzymes in the early part of the pathway leading to the sesquiterpenoid phytoalexin ipomeamarone. A recent paper by Gustine et al...

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ABSTRACICasbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.