Competitive inhibition of aristolochene synthase by phenyl-substituted farnesyl diphosphates: evidence of active site plasticity

Analogues of farnesyl diphosphate (FPP, 1) containing phenyl substituents in place of methyl groups have been prepared in syntheses that feature use of a Suzuki-Miyaura reaction as a key step. These analogues were found not to act as substrates of the sesquiterpene cyclase aristolochene synthase from Penicillium roqueforti ( AS). However, they were potent competitive inhibitors of AS with K-I-values ranging from 0.8 to 1.2 mu M. These results indicate that the diphosphate group contributes the largest part to the binding of the substrate to AS and that the active sites of terpene synthases are sufficiently flexible to accommodate even substrate analogues with large substituents suggesting a potential way for the generation of non-natural terpenoids. Molecular mechanics simulations of the enzyme bound inhibitors suggested that small changes in orientations of active site residues and subtle alterations of the conformation of the backbones of the inhibitors are sufficient to accommodate the phenyl-farnesyl-diphosphates.