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Presilphiperfolan-8beta-ol synthase, encoded by the BcBOT2 gene from the necrotrophic plant pathogen Botrytis cinerea, catalyzes the multistep cyclization of farnesyl diphosphate (2) to the tricyclic sesquiterpene alcohol presilphiperfolan-8beta-ol (3), the preursor of the phytotoxin botrydial, a strain-dependent fungal virulence factor. Incubation of (1R)-1-(2)Hfarnesyl diphosphate (2b) with recombinant presilphiperfolan-8beta-ol synthase gave exclusively (5R)-5alpha-(2)H-3b, while complementary incubation of (1S)-1-(2)HFPP (2c) gave (5S)-5beta-(2)H-3c. These results established that cyclization of farnesyl diphosphate involves displacement of the diphosphate group from C-1 with net inversion of configuration and ruled out the proposed intermediacy of the cisoid conformer of nerolidyl diphosphate (9) in the cyclization. While not a mandatory intermediate, (3R)-nerolidyl diphosphate was shown to act as a substrate surrogate. Cyclization of 13,13,13-(2)H(3) farnesyl diphosphate (2d) gave 14,14,14-(2)H(3)-3d, thereby establishing that electrophilic attack takes place exclusively on the si face of the 12,13-double bond of 2. The combined results provide a detailed picture of the conformation of enzyme-bound farnesyl diphosphate at the active site of presilphiperfolan-8beta-ol synthase.
Wang et al. (Thu,) studied this question.