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

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Abstract
Mevalonate-14C was incorporated into (—)-kaurene-14C in cell-free extracts of immature pea (Pisum sativum L.) seeds. The identification of 14C-product as (—)-kaurene was based on: A) comparison with authentic (—)-kaurene on thin-layer and gas-liquid chromatography; and B) oxidation of 14C-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 Mg2+ or Mn2-, with Mn2+ being a more effective activator than Mg2+. 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.