Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog

Abstract

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 the proper positioning of carbons C1 and C6 to enable cyclization. However, because of 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 the cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene, 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 of LPP as an intermediate.

Fig. 1.. Chemical mechanism proposed for the enzymatic formation of (+)-limonene from geranyl diphosphate (GPP).

Fig. 2.. Chemical structures of 2-fluorogeranyl diphosphate (FGPP), 8,9-difluorogeranyl diphosphate (DFGPP), 8,9-difluorolinalyl diphosphate (DFLPP), and 6,7-dihydrogeranyl diphosphate (6,7-dihydroGPP).

Fig. 3.. Rapid mixing/quench flow measurements for reaction of (+)-LS with GPP and LPP substrates.

200 μM enzyme was rapidly mixed with an equal volume of 2 mM substrate (GPP, circles or LPP, triangles), and the reaction quenched after the indicated incubation times by mixing with 2N KOH followed by injection of the sample into hexanes for extraction of the limonene product. Limonene concentration was then determined by GC-MS. Error bars from duplicate experiments.

Fig. 4.. Production of DFM in the reaction of (+)-LS with DFGPP.

A, GC-MS analysis of product generated in the reaction of WT (+)-LS with DFGPP in red compared to standard limonene control in black. B, Fragmentation pattern for the new product at retention time 7.65 minutes with a parent ion of 172 m/z (limonene is 136).

Fig. 5.. NMR confirmation of the DFM product.

A, Chemical structure of the novel product DFM. B, proton NMR spectrum for the product (DFM) from the reaction of (+)-LS with DFGPP. Chemical shifts reported in ppm relative to TMSP. An expanded spectrum is presented in Fig. S1. C, Fluorine NMR spectrum for the product (DFM) from the reaction of (+)-LS with DFGPP. Chemical shifts reported in ppm relative to NaF. Both NMR spectra were recorded with sample in deuterated benzene.

Fig. 6.. Proposed mechanism for the formation of DFM from DFLPP via general base catalyzed loss of a methyl proton at C10.

Fig. 7.. Active-site electron density shown in wall-eyed stereoview for ligand and metal ions following a 30 min soak of (+)-LS with DFGPP and MnCl₂.

The figure shows two superimposed Polder maps calculated individually for 3 Mn²⁺ ions (at 13 σ cutoff colored in red) and for the DFLPP ligand (at 4 σ cutoff colored in cyan). Coordinating solvent molecules have not been modeled into the electron density surrounding the metal ions.

Fig. 8.. Superposition of active sites for (+)-LS and BPPS showing similar extended conformations of the FGPP (yellow) and 3-aza-2,3 dihydrogeranyl diphosphate (salmon) ligands following alignment of the protein chains from (+)-LS and BPPS, respectively.

Phosphorous, orange; oxygen, red; nitrogen, blue; and fluorine, silver.

Fig. 9.. Ligands in the active site of (+)-LS showing similar extended conformations of FGPP and DFLPP following soaks with FGPP (yellow) and DFGPP (green).

A, superposition of FGPP and DFGPP after alignment of the protein chains in the two structures. B, side-by-side presentation of the two ligands to more clearly visualize how syn migration of the diphosphate in DFGPP would lead to the R-enantiomer of DFLPP. Phosphorous, orange; oxygen, red; and fluorine, silver.