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

Abstract

ABSTRACIFarnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg2" ion at 4 millimolar gave the greatest stimulation of activity; Mn2 ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (Km = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K,,, = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K. = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes-a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.Casbene, a macrocyclic diterpene hydrocarbon with antifungal antibiotic properties, is produced in cell-free extracts of castor bean seedlings which have been exposed to spores of Rhizopus stolonifer or other fungi (20), to the extracellular endopolygalacturonase of R. stolonifer culture filtrates (12), or to pectic fragments released from pectic substances of the plant cell wall

An In Vitro Assay for Monitoring Prenyl Transferase Activity in Lepidopteran Corpora Allata

“…Although significant cytosolic FPP synthase activity can be detected in homogenates of decapitated M. sexta larvae whose guts were removed, only small amounts of FPP formation are detected in corpora allata homogenates when conventional assay buffers (20-100 mM Tris-HCl or phosphate buffer, pH 7, 2-5 mM MgCl 2 , containing 0-10 mM KF to inhibit endogenous phosphatases) are used. Similar low levels of short-chain prenyl transferase activity have been noted in plants and bacteria and have been attributed to either enzyme lability or membrane association (Green et al, 1975;Takahashi and Ogura, 1981;Spurgeon et al, 1984;Sagami and Ogura, 1985;Dudley et al, 1986;Heide and Berger, 1989;Croteau and Purkett, 1989). Because activity in these cases was increased by the addition of glycerol and we decided to examine the effect of several additives on corpora allata prenyl transferase activity.…”

Reverse Transcription-PCR analysis of geranylgeranyl diphosphate synthase (JcGGPPS) in Jatropha curcas L. and in silico analysis of Casbene Synthase (JcCS) among Euphorbiaceae

“…The GGPPS is a homodimeric prenyl transferase occurred ubiquitously in plants, animals and bacteria [8]. Previous studies showed that heterologous expression of GGPPS gene in Escherichia coli or Saccharomyces cerevisiae resulted in the accumulation of diterpenes, including casbene [9]. This finding indicates that a large increase in GGPPS activity might correspond to the accumulation of diterpenes.…”

Reducing of phorbol ester content in callus cultures of physic nut (Jatropha Curcas L.) using manganese chloride and n-ethylmaleimid

“…Meanwhile, at a concentration of more than 1 mM Mn 2+ are strongly inhibitors. [11] Another study mentioned that a high concentration of Mn 2+ disrupt the bond between the substrate with the enzyme. [12] The treatment was also carried out with n-etilmaeimid (NEM) on brownish callus.…”

“…Based on the literature, NEM can inhibite IPP isomerase enzyme, geranil transferase, and farnesil transferase activity. [10,11] Since both compounds were found to have influence in the biosynthesis secondary metabolites through IPP and DMAPP pathway, they were chosen as inhibitor of phorbol esters. These toxic compounds belonging to diterpen were synthesized via IPP and DMAPP pathway.…”