Structure and reactivity of the dammarenyl cation: configurational transmission in triterpene synthesis

The dammarenyl cation is the last common intermediate in the cyclization of oxidosqualene to a diverse array of secondary triterpene metabolites in plants. We studied the structure and reactivity of the dammarenyl cation to understand the factors governing the regio- and stereospecificity of triterpene synthesis. First, we demonstrated that the dammarenyl cation has a 17-alpha side chain in Arabidopsis thaliana lupeol synthase (LUP1) by incubating the substrate analogue (18E)-22,23-dihydro-20-oxaoxidosqualene with LUP1 from a recombinant yeast strain devoid of other cyclases and showing that the sole product of (18E)-22,23-dihydro-20-oxaoxidosqualene was 3-alpha-hydroxy-22,23,24,25,26,27-hexanor-17-alpha-dammaran-20-one. Quantum mechanical calculations were carried out on gas-phase models to show that the 20-oxa substitution has negligible effect on substrate binding and on the activation energies of reactions leading to either C17 epimer. Further molecular modeling indicated that, because of limited rotational freedom in the cyclase active site cavity, the C17 configuration of the tetracyclic intermediate can be deduced from the angular methyl configuration of the pentacyclic or 6-6-6-6 tetracyclic product. This rule of configurational transmission aided in elucidating the mechanistic pathway accessed by individual cyclases. Grouping of cyclases according to mechanistic and taxonomic criteria suggested that the transition between pathways involving 17-R and 17-alpha intermediates occurred rarely in evolutionary history. Two other mechanistic changes were also rare, whereas variations on cation rearrangements evolved readily. This perspective furnished insights into the phylogenetic relationships of triterpene synthases.