Pinene cyclase

As is indicated in Fig. 22-3, the same intermediate cation can yield a variety of end products. For example, pure geranyl diphosphate pinene cyclase catalyzes formation of several other terpenes in addition to a-pinene.89 Another aspect of terpene synthesis is that insects may convert a plant terpene into new compounds for their own use. For example, myrcene, which is present in pine trees, is converted by bark beetles to ipsenol (Fig. 22-3), a compound that acts as an aggregation pheromone.90... 

Two pinene cyclases have been isolated from sage (19,35). Electrophoretically pure pinene cyclase I converts geranyl pyrophosphate to (+)-a-pinene and to lesser quantities of (+)-camphene and (+)-limonene, whereas pinene cyclase II, of lower molecular weight, converts the acyclic precursor to (-)-B-pinene and to lesser quantities of (-)-a-pinene, (-)-camphene and (-)-limonene. Both purified enzymes also utilize neryl and linalyl pyrophosphate as alternate substrates for olefin synthesis. The availability of enzyme systems catalyzing formation of enantiomeric products from a common, achiral substrate has provided an unusual opportunity to examine the stereochemistry of cyclization. 

The pinene cyclases convert the anomalous linalyl enantiomer to abnormal levels of acyclic (e.g. myrcene) and monocyclic (e.g. limonene) terpenes, these aberrant products perhaps arising via ionization of the tertiary substrate in the transoid or other partially extended (exo) form (see below). In any event, for all "normal" cyclizations examined thus far, the configuration of the cyclizlng linalyl Intermediate has been confirmed to be that which would be expected on the basis of an anti-endo conformation. Scattered attempts at intercalating the cyclization cascade with analogs of proposed cyclic intermediates (e.g. a-terpinyl and 2-pinyl pyrophosphate) have been unsuccessful (20,35,36). 

Figure 6. Conversion of 6,7-dihydrogeranyl pyrophosphate, via 6,7-dihydrolinalyl pyrophosphate, to dihydro olefins and alcohols by (+)-bornyl pyrophosphate and (+)-pinene cyclases.

In a subsequent examination of the cyclases that formed ( 4-)- and ( — )-pinene, cyclase 1,96,000 MW, converted geranyl pyrophosphate to (-1-)-a-pinene (d-a-pimm) (14), but produced smaller amounts of (-b)- or if-camphene (15) and (+)- or /-limonene (11) as side products (Johnson and Croteau, 1987). Cyclase II, 55,000 MW, transformed geranyl pyrophosphate into ( —)-p-pinene (/- -pinene) (16) along with smaller amounts of /-a- or (— )-a-pinene, (-)- or /-camphene, ( —)- or /-limonene, and myrcene (17) as coproducts (Fig. 19.7) (Croteau, 1984 Johnson and Croteau, 1987). Extensive purification of each enzyme and differential inactivation studies ensured that each set of stereochemi-cally related products was synthesized by a single, distinct enzyme. 

Cyclases responsible for the formation of (+)-a-pinene and (+)-llmonene, monoterpenes which inhibit fungal growth and repel bark beetles, have been studied in herbaceous species. Two distinct types of enzymes synthesize antipodal monoterpene hydrocarbons. The differential expression of the cyclases responsible for co-productlon of enantiomeric monoterpenes may determine the highly selective resistance response conifers exhibit toward bark beetles and their fungal symbionts. 

More than a dozen cDNAs encoding monoterpene synthases from a range of plant sources have now been isolated [22, 32-36]. The corresponding recombinant enzymes generate a diverse set of products and provide an exceptional set of catalysts for the detailed study of structure-function relationships in monoterpene cyclization. However, it was earher work with native synthases that first allowed definition of the cyclization reaction [ 15]. Thus, the stereochemical model for monoterpene cyclization was developed largely from studies with the bicyclic monoterpene cyclases (-i-)- and (-)-bornyl diphosphate synthases, (-1-)-and (-)-pinene synthases, and (-)-e do-fenchol synthase. It was also the purification of several native monoterpene synthases that gave the first indication that... 

The labeling patterns of antipodal bornane and plnane monoterpenes derived from [l- H]geranyl pyrophosphate by partially purified cyclases have recently been determined [(+)- and (-)-bornyl pyrophosphate are labeled at C3, and (+)- and (-)-pinenes at C7].22 2ii These results indicate that the antipodes are derived via enantiomeric cyclizations Involving antipodal linalyl and a-terpinyl intermediates, rather than by... 

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