Biosynthesis of Monoterpenes and Sesquiterpenes

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Monoterpenes and sesquiterpenes are the C10 and C15 members of the isoprenoid family of plant products. Selective techniques for extraction of glandular trichomes and affinity chromatography methods allow purification of the enzymes of terpene metabolism in high yield. This research tests a stereochemical model for the coupled isomerization- cyclization of geranyl pyrophosphate to symmetrical monoterpenes. Stereospecifically labeled precursors, selective chemical degradation schemes and chromatographic resolution of enantiomers, permit the cryptic stereochemistries of the reaction steps, and hydride-shift patterns, to be deciphered. Four types of sesquiterpene cyclases are being used to compare the mechanism, stereochemistry and enzymology of sesquiterpene biosynthesis. Cytochrome P450-dependent hydroxylases catalyze the first step in the oxidative transformation of parent cyclic terpenoids; and several of these highly specific mixed- function oxidases have been isolated. These microsomal systems will be solubilized and separated into components, and reconstitution experiments, kinetic analysis, and binding studies will be employed to establish the basis for the extreme substrate selectivity observed. Potent, highly selective inhibitors of the hydroxylases will be designed which, along with a specific inhibitor of cyclization, will be used to manipulate terpene production in vivo. %%% Many biological molecules and natural products are composed of a basic five-carbon building block. Terpenes are the compounds made from this building block that impart fragrances to many plants making them useful as perfume, spices and attractants or repellants to insects. This research will improve our understanding of terpene metabolism, aid in attempts to exploit ecological interactions involving terpenoids, and assist in efforts to improve production of these commercially valuable natural products.