Biosynthesis of gibberellins. IV. Biosynthesis of cyclic diterpenes from trans-geranylgeranyl pyrophosphate.

A l*C-diterpene alcohol (copalol) was isolated as its 4-(4’nitrophenylazo)benzoyl ester from a soluble enzyme system which had been prepared from a cell-free extract of mycelia of the fungus Gibberella fujikuroi and incubated with ZJ*Cmevalonate, ATP, and MgC12. Copalol was identified as trans-( -)-labda-8(16), 13-dien-15-01 from its properties, in comparison with those of the enantiomeric alcohol synthesized from manool. 14C-Geranylgeraniol and l*C-( -)kaurene were also identified as diterpenes formed in these incubation mixtures. All three diterpenes were also found as products when frans J*C-geranylgeranyl pyrophosphate was used as the substrate in place of EJ4C-mevalonate and ATP. A substance with the properties expected for 14C-copalyl pyrophosphate was also extracted with collidine from these incubation mixtures with either ZJ*C-mevalonate or fransl*C-geranylgeranyl pyrophosphate as the substrate. The structure of the purified product was established as copalyl pyrophosphate by showing (a) that it yielded copalol on treatment with alkaline phosphatase and (b) that the doubly labeled product was formed from fransJ*C-geranylgeranyl P2P-pyrophosphate with the same W:azP ratio as the substrate. Copalyl pyrophosphate was converted to kaurene by a soluble enzyme preparation from G. fujikuroi. The plant growth retardant tributyl-2,4-dichlorobenzylphosphonium chloride (Phosfon) at 1 mru concentration inhibited this conversion, whereas two other retardants, /?-chloroethyltrhnethylammonium chloride (CCC) and 2’-isopropyl-4’-(trimethylammonium chloride)-5’-methylphenyl piperidine carboxylate (Amo 1618), at the same concentration were not significantly inhibitory. All three retardants inhibit the over-all conversion of fronsJ*C-geranylgeranyl pyrophosphate to kaurene in this system. Copalyl pyrophosphate was also converted to ( -)-kaurene in a soluble enzyme fraction from the endosperm of wild cucumber (Echinocystis mucroca7pa) seed and to a mixture of ( -)-kaurene, (+)-stachene, (+)-sandaracopimaradiene, and trachylobane in a soluble enzyme fraction prepared from seedlings of castor bean (Ricinus communis).