Biosynthesis of monoterpenes: Demonstration of a geranyl pyrophosphate:(−)-bornyl pyrophosphate cyclase in soluble enzyme preparations from tansy ( Tanacetum vulgare)

Abstract

Tansy ( Tanacetum vulgare L.) produces an essential oil containing the optically pure monoterpene ketone, (−)-camphor, as a major constituent. A soluble enzyme preparation from immature leaves of this plant converts the acyclic precursor [1- 3H]geranyl pyrophosphate to the bicyclic monoterpene alcohol borneol in the presence of MgCl 2, and oxidizes a portion of the borneol to camphor in the presence of a pyridine nucleotide. The identity of the major biosynthetic product as borneol was confirmed by chemical oxidation to camphor and crystallization of the derived oxime to constant specific radioactivity. The stereochemistry of the borneol was verified as the (−)-(1 S,4 S) isomer by oxidation to camphor, conversion to the corresponding ketal with D-(−)-2,3-butanediol, and separation of diastereoisomers by radio-gas-liquid chromatography. When enzyme reaction mixtures were treated with a mixture of acid phosphatase and apyrase, following an initial ether extraction of labeled borneol, additional quantities of borneol were generated, indicating the presence of a phosphorylated derivative of borneol. This water-soluble metabolite was prepared by large-scale enzyme incubations with [1- 3H]geranyl pyrophosphate (plus phosphatase inhibitor), and the identity of the initial cyclization product was established as (−)-bornyl pyrophosphate by direct ion-exchange chromatographic analysis and enzymatic hydrolysis. The pathway for the formation of (−)-(1 S,4 S)-camphor was therefore identical to that previously demonstrated for the (+)-(1 R,4 R) isomer, involving cyclization of geranyl pyrophosphate to bornyl pyrophosphate, hydrolysis of this intermediate to borneol, and oxidation of the alcohol to the ketone. The labeling pattern of the product derived from [1- 3H 2, U- 14C]geranyl pyrophosphate was determined by oxidation of the biosynthetic borneol to camphor and selective removal of tritium by exchange of the α hydrogens at C3 of the ketone. This labeling pattern was identical to that observed previously for the (+) isomer, suggesting the same mechanism of cyclization, but of opposite enantiospecificity. Some properties of the antipodal (+)- and (−)-bornyl pyrophosphate cyclases were compared.