Biosynthesis of (-)-Kaurene in Cell-free Extracts of Immature Pea Seeds

Abstract

Mevalonate-(14)C was incorporated into (-)-kaurene-(14)C in cell-free extracts of immature pea (Pisum sativum L.) seeds. The identification of (14)C-product as (-)-kaurene was based on: A) comparison with authentic (-)-kaurene on thin-layer and gas-liquid chromatography; and B) oxidation of (14)C-product and (-)-kaurene with osmium tetroxide to form the common derivative kaurane-16,17-diol. The enzyme system is heat labile and is dependent upon ATP and Mg(2+) or Mn(2-), with Mn(2+) being a more effective activator than Mg(2+). The reaction rate was proportional to enzyme concentration in reaction mixtures containing 0.45 to 1.8 mg protein n/ml, and was linear with time through 120 minutes in standard reaction mixtures. Enzyme preparations from immature seeds of tall and dwarf peas appeared to synthesize (-)-kaurene at the same rate. Synthesis of (-)-kaurene was readily inhibited by Amo-1618. (2-Chloroethyl)-trimethylammonium chloride (CCC) also inhibited (-)-kaurene synthesis; however, approximately 1000-fold higher concentrations of CCC were required to evoke the same percentages of inhibition as Amo-1618.