Biosynthesis of Monoterpenes: Regio- and Stereochemistry of (+)-3-Carene Biosynthesis

Incubation of [1-3H1]geraniol with stem disks of Douglas fir (Pseudotsuga menziesii) and incubation of [1-3H1]geranyl pyrophosphate with both a soluble enzyme extract from Douglas fir and a partially purified preparation of (+)-3-carene synthase from lodgepole pine (Pinus contorta) resulted in the production of (+)-3-[3H] carene. Subsequent conversion of the product to car-3-en-5-one and to 4-isocaranone followed by base-catalyzed exchange of the α-hydrogens established that the 3H located at C1 in the geranyl substrate resided at C5 of (+)-3-carene. Incubation of the (+)-3-carene synthase preparation with (S)-[5-3H1, 4-14C]geranyl pyrophosphate resulted in the production of (+)-3-carene without loss of tritium, indicating that the 5-proR hydrogen is eliminated during cyclopropyl ring closure. Analysis of the conformational requirements for this 1,3 elimination involving the 5-proR hydrogen suggested that cyclopropyl ring formation occurs via a (4S)-α-terpinyl cation derived from the anti-endo cyclization of a (3S)-linalyl pyrophosphate intermediate. Kinetic analyses of the conversion of (1Z, 3R)-[1-3H1]linalyl pyrophosphate, (1Z, 3S)-[1-3H1]linalyl pyrophosphate and [1-3H1]geranyl pyrophosphate by (+)-3-carene synthase revealed that the velocity of the reaction with the (3S)-linalyl enantiomer was 25-fold greater than the velocity with the (3R)-enantiomer and twice that of the natural substrate, geranyl pyrophosphate, thereby confirming this stereo-chemical prediction and also indicating that the cyclization of the linalyl intermediate is faster than the coupled isomerization and cyclization of the geranyl substrate. From these results, a model that details the regio- and stereochemistry of the enzymatic conversion of geranyl pyrophosphate to (+)-3-carene is proposed.