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 monoterpene ether 1,8-cineole (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane) by a process thought to involve the initial isomerization of the substrate to the tertiary allylic isomer, linalyl pyrophosphate, and cyclization of this bound intermediate to the alpha-terpinyl carbocation that is subsequently captured by water and undergoes heterocyclization to the remaining double bond. The enzyme was isolated from the secretory cells of the glandular trichomes of Salvia officinalis (garden sage) and partially purified, and the properties of this monoterpene cyclase, previously determined in crude cell-free extracts, were reexamined. These properties (pH optimum, divalent metal ion requirement, molecular weight, pI) were similar to those determined previously with the exception of substrate utilization; geranyl pyrophosphate was shown to be a more efficient substrate than the cis-isomer, neryl pyrophosphate, in the absence of competing phosphatase activity that contaminated earlier preparations of this enzyme. As with other monoterpene cyclases of herbaceous species, cineole synthase was inhibited by cysteine- and histidine-directed reagents, and protection against inactivation was provided by the substrate-metal ion complex. Studies with 18O-labeled acyclic precursors and H(2)18O, followed by mass spectrometric analysis of the product, confirmed that water was the sole source of the ether oxygen atom of 1,8-cineole. The electrophilic nature of the coupled isomerization-cyclization reaction was examined with a series of substrate and intermediate analogues. The overall stereochemistry of the cyclization of geranyl pyrophosphate to the symmetrical monoterpene was established by determining the enantioselectivity for (3R)- or (3S)-linalyl pyrophosphate as an alternative substrate and by oxidation of [3-3H]1,8-cineole, derived from [1-3H]geranyl pyrophosphate, to (+/-)-3-keto-1,8-cineole and radio-GLC separation of diastereomeric ketal derivatives to determine the labeled enantiomer.
Croteau
R
R
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-6340.
Alonso
W R
WR
Koepp
A E
AE
Johnson
M A
MA
D019755:Carbon-Carbon Lyases; D002845:Chromatography; D003500:Cyclization; D003511:Cyclohexanols; D000077591:Eucalyptol; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008190:Lyases / Q000302:isolation & purification* / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D008610:Menthol / Q000031:analogs & derivatives / Q000378:metabolism; D008968:Molecular Conformation; D008970:Molecular Weight; D039821:Monoterpenes; D010100:Oxygen / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013729:Terpenes; D014867:Water / Q000378:metabolism
D003511:Cyclohexanols; D039821:Monoterpenes; D011106:Polyisoprenyl Phosphates; D013729:Terpenes; D014867:Water; D008610:Menthol; D008345:Manganese; C015234:geranyl pyrophosphate; D008190:Lyases; C019899:1,8-cineole synthetase; D019755:Carbon-Carbon Lyases; D000077591:Eucalyptol; D010100:Oxygen