Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of linalyl pyrophosphate to (-)-endo-fenchol.

Abstract

The conversion of geranyl pyrophosphate to (-)-endo-fenchol is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. To test this stereochemical scheme, phosphatase-free preparations of (-)-endo-fenchol cyclase from fennel (Foeniculum vulgare M.) fruit were repeatedly incubated with a sample of (3RS)-[1-3H2]linalyl pyrophosphate until approximately 50% of this precursor was converted to the bicyclic monoterpenol end product. The residual linalyl pyrophosphate was isolated and enzymatically hydrolyzed to the free alcohol, linalool, which was resolved by chiral phase capillary gas-liquid chromatography of the derived threo and erythro mixture of 1,2-epoxides. The predominance of the (3S)-enantiomer in the residual substrate indicated that the (3R)-enantiomer was preferred for the cyclization to (-)-(1S)-endo-fenchol. This conclusion was subsequently confirmed by the preparation and direct testing of (3R)-1Z-[1-3H] linalyl pyrophosphate, which afforded a Km value lower than that observed for geranyl pyrophosphate and a relative velocity nearly three times higher. (3S)-1Z-[1-3H]Linalyl pyrophosphate was not an effective substrate for (-)-endo-fenchol biosynthesis but did, by an anomalous cyclization, give rise to low levels of the enantiomeric (+)-(1R)-endo-fenchol as well as to other products. These results support the proposed stereochemical model and also suggest that the isomerization step is rate limiting in the coupled isomerization-cyclization of geranyl pyrophosphate to (-)-endo-fenchol.