In vitro biosynthesis of cadinanes by cell-free extracts of cultured cells of Heteroscyphus planus

Abstract

A cell-free extract from the calli of the liverwort Heteroscyphus planus catalyzes the divalent metal ion-dependent conversion of (2Z, 6E)-farnesyl diphosphate (FPP) into (-)-γ-cadinene and (+)-germacrene D, while it specifically converts (2E, 6E)-FPP into (+)-cubenene and (+)-epicubenol. The 1,3-hydride shift in the formation of (-)-γ-cadinene has been determined by conversion of (2Z, 6E)-[1,1-²H₂]-FPP into (-)-γ-cadinene which was shown by GLC–MS and ²H NMR spectroscopy to be labelled at the C-11 position. These findings suggest that (-)-γ-cadinene is directly formed from 2Z, 6E-FPP by intramolecular electrophilic attack of the primary carbocation on the C-10 position of FPP. (+)-Cubenene synthase and (-)-γ-cadinene synthase has been purified by fractionation with ammonium sulfate, gel filtration on Sephacryl S-200 HR and anion exchange chromatography on DEAE-Sepharose CL-6B. Separation of (-)-γ-cadinene synthase from (+)-cubenene synthase is facilitated by a change in the elution behaviour of enzymes during anion exchange chromatography. All the evidence strongly suggests that the activities of (+)-cubenene synthase and (+)-epicubenol synthase are dual functions of the same enzyme.

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