BIOSYNTHESIS OF MONOTERPENES - PARTIAL-PURIFICATION, CHARACTERIZATION, AND MECHANISM OF ACTION OF 1,8-CINEOLE SYNTHASE

Geranyl pyrophosphate:1,8-cineole cyclase (cineole synthase) catalyzes the conversion of geranyl pyrophosphate to the symmetrical monoterpen e ether 1,8-cineole (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane) by a p rocess thought to involve the initial isomerization of the substrate t o the tertiary allylic isomer, linalyl pyrophosphate, and cyclization of this bound intermediate to the alpha-terpinyl carbocation that is s ubsequently captured by water and undergoes heterocyclization to the r emaining double bond. The enzyme was isolated from the secretory cells of the glandular trichomes of Salvia officinalis (garden sage) and pa rtially purified, and the properties of this monoterpene cyclase, prev iously determined in crude cell-free extracts, were reexamined. These properties (pH optimum, divalent metal ion requirement, molecular weig ht, pI) were similar to those determined previously with the exception of substrate utilization; geranyl pyrophosphate was shown to be a mor e efficient substrate than the cis-isomer, neryl pyrophosphate, in the absence of competing phosphatase activity that contaminated earlier p reparations of this enzyme. As with other monoterpene cyclases of herb aceous species, cineole synthase was inhibited by cysteine- and histid ine-directed reagents, and protection against inactivation was provide d by the substrate-metal ion complex. Studies with O-18-labeled acycli c precursors and (H2O)-O-18, followed by mass spectrometric analysis o f the product, confirmed that water was the sole source of the ether o xygen atom of 1,8-cineole. The electrophilic nature of the coupled iso merization-cyclization reaction was examined with a series of substrat e and intermediate analogues. The overall stereochemistry of the cycli zation of geranyl pyrophosphate to the symmetrical monoterpene was est ablished by determining the enantioselectivity for (3R)- or (3S)-linal yl pyrophosphate as an alternative substrate and by oxidation of [3-H- 3]1,8-cineole, derived from [1-H-3]geranyl pyrophosphate, to (+/-)-3-k eto-1,8-cineole and radio-GLC separation of diastereomeric ketal deriv atives to determine the labeled enantiomer. (C) 1993 Academic Press, I nc.