Biosynthesis of the sesquiterpene botrydial in Botrytis cinerea. Mechanism and stereochemistry of the enzymatic formation of presilphiperfolan-8beta-ol

Abstract

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)H]farnesyl diphosphate (2b) with recombinant presilphiperfolan-8beta-ol synthase gave exclusively (5R)-[5alpha-(2)H]-3b, while complementary incubation of (1S)-[1-(2)H]FPP (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.

Figure 1

Key A) HMBC and B) NOESY correlations for 3.

Scheme 1

Biosynthesis of Botrydial (1).

Scheme 2

Cyclization of FPP (2) and of the surrogate substrate (3R)-NPP (9) to 3.