Biosynthesis of the Diterpene Phytoalexin Casbene: Partial Purification and Characterization of Casbene Synthetase from Ricinis communis

Abstract

Casbene synthetase from 67-hour seedlings of Ricinis communis L. which had been treated with Rhizopus stolonifer spores was purified 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 partially purified enzyme exhibited a broad pH optimum from pH 7.5 to 9.0 with half-maximal activity at pH 6.0 and 9.8. Chromatography on a calibrated Sephadex G-100 column indicated a molecular weight of 53,000 +/- 3,000 for casbene synthetase. Concentrations of Mg(2+) above 5 mm gave maximal stimulation of the activity. Mn(2+) was much less effective and was inhibitory at concentrations above 0.2 mm. The K(m) for geranyl-geranyl pyrophosphate was estimated as 1.9 mum. The activity was inhibited 50% by 2.5 mm N-ethylmaleimide; 10 mm iodoacetamide was not inhibitory. N,N-Dimethylaminoethyl-2,2-diphenylpentanoate (SKF-525A) and the growth retardant 2'isopropyl-4'-(trimethylammonium chloride)-5'-methylphenyl piperidine-1-carboxylate (Amo-1618) were ineffective inhibitors of casbene synthetase, but the growth retardant tributyl-2, 4-dichlorobenzyl-phosphonium chloride (Phosphon D) at a concentration of 5 mum inhibited the activity by 55%.