Biosynthesis of the Diterpene Phytoalexin Casbene

Abstract

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...

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings

“…This result is expected since the results of Table II Earlier work had demonstrated that casbene synthetase activity increased substantially after elicitation in comparison with the undetectable or very low values found in extracts of uninfected controls (7,28). The experiments summarized in Table I indicate that elicitation of casbene synthesis in 3-d-old castor bean seedlings results not only in an increase in casbene synthetase activity, but also in the activities of geranyl transferase and farnesyl transferase.…”

“…The postulated intermediates and enzymes involved in the biosynthesis of casbene from MVA are illustrated in Figure 1. Two geranyl transferase isoenzymes (11), farnesyl transferase (6), and casbene synthetase (7) Mg protein/ml). Preliminary studies indicated that the reaction rate was linear with this range of enzyme concentrations for 5 min at 30°C.…”

“…Casbene synthetase activity was measured in the crude 27,000g or 37,000g supematant fraction with GGPP as the substrate under assay conditions previously described (7).…”

Purification and Characterization of an Inducible Sesquiterpene Cyclase from Elicitor-Treated Tobacco Cell Suspension Cultures

“…Croteau et al (12) recently reported that the accumulation of mono-and diterpenes by lodgepole pine saplings infected with Ceratocystis clavigera was correlated with an induction of mono-and diterpene cyclases. Similarly, the induction of casbene synthetase, a diterpene cyclase, has been correlated with the accumulation of the diterpene phytoalexin casbene by castor bean seedlings infected with Rhizopus stolonifer (14). Moesta and West (24) have further demonstrated that the increase in casbene synthetase enzyme activity was preceded by a transient induction ofthe casbene synthetase mRNA translational activity, and they suggested that casbene synthetase was under transcriptional control.…”

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings

“…Polyclar AT was not included in the homogenization medium despite its use for the isolation of other enzymes from castor bean (6). At the lower pH values employed in this extraction procedure the interactions of phenolic substances and proteins are considerably reduced, thus eliminating the necessity for Polyclar AT.…”