(-GERMACRENE-A BIOSYNTHESIS - THE COMMITTED STEP IN THE BIOSYNTHESIS OF BITTER SESQUITERPENE LACTONES IN CHICORY())

The leaves and especially the roots of chicory (Cichorium intybos L..) contain high concentrations of bitter sesquiterpene lactones such as the guianolides lactupicrin, lactucin, and 8-deoxylactucin. Eudesmanol ides and germacranolides are present in smaller amounts. Their postula ted biosynthesis through the mevalonate-farnesyl diphosphate-germacrad iene pathway has now been confirmed by the isolation of a (+)-germacre ne A synthase from chicory roots. This sesquiterpene cyclase was purif ied 200-fold using a combination of anion-exchange and dye-ligand chro matography. It has a K-m value of 6.6 mu M, an estimated molecular mas s of 54 kD, and a (broad) pH optimum around 6.7. Germacrene A, the enz ymatic product, proved to be much more stable than reported in literat ure. Its heat-induced Cope rearrangement into (-)-beta-elemene was uti lized to determine its absolute configuration on an enantioselective g as chromatography column. To our knowledge, until now in sesquiterpene biosynthesis, germacrene A has only been reported as an (postulated) enzyme-bound intermediate, which, instead of being released, is subjec ted to additional cyclization(s) by the same enzyme that generated it from farnesyl diphosphate. However, in chicory germacrene A is release d from the sesquiterpene cyclase. Apparently, subsequent oxidations an d/or glucosylation of the germacrane skeleton, together with a germacr ene cyclase, determine whether guaiane- or eudesmane-type sesquiterpen e lactones are produced.