Mechanism of monoterpene cyclization: Stereochemistry of the transformation of noncyclizable substrate analogs by recombinant (-)-limonene synthase, (+)-bornyl diphosphate synthase, and (-)-pinene synthase

All monoterpene cyclases investigated to date are capable of overcoming the topological impediment to direct cyclization of the universal, acyclic C10 intermediate of isoprenoid biosynthesis geranyl diphosphate. Although strong suggestive evidence has been accumulated for the intermediary linalyl diphosphate in cyclase catalysis, all previous efforts to directly observe this product at the mandatory isomerization step have failed. (-)-4S-Limonene synthase from spearmint (Mentha spicata), (+)-bornyl diphosphate synthase from sage (Salvia officinalis), and (-)-pinene synthase from grand fir (Abies grandis) have been expressed in Escherichia coli and the recombinant enzymes have been isolated and purified. These enzymes were examined with the noncyclizable substrate analogs 6,7-dihydrogeranyl diphosphate and 2,3-methanogeranyl diphosphate to gain insight into the normally cryptic isomerization step of the reaction sequence. The analogs were catalytically active, affording acyclic olefins and alcohols as products. Chiral phase gas chromatography and mass spectrometry analysis provided evidence that the normal cyclization of geranyl diphosphate by (-)-4S-limonene synthase and by (-)-pinene synthase proceeds via preliminary isomerization to the bound tertiary intermediate 3S-linalyl diphosphate, whereas the cyclization catalyzed by (+)-bornyl diphosphate synthase proceeds via the intermediate 3R-linalyl diphosphate.