Direct evidence for an enzyme generated LPP intermediate in (+)-limonene synthase using a fluorinated GPP substrate analog.

Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans-C2-C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes proper positioning of carbons C1 and C6 to enable cyclization. However, due to the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP, despite significant efforts. Here, we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP) which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP), while preventing the subsequent ionization/cyclization to produce the α-terpinyl cation. Steady state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene (DFM), is produced. When crystals of apo-LS are soaked with DFGPP under conditions limiting turnover of the enzyme, we show using x-ray crystallography that DFLPP is produced in the enzyme active site and trapped in the crystals. Clear electron density is observed in the active site of the enzyme but it cannot be appropriately fit with a model for the DFGPP substrate analog whereas it can accommodate an extended conformation of DFLPP. This result supports the current model for monoterpene cyclization by providing direct evidence for LPP as an intermediate.