Intramolecular proton transfer in the cyclization of geranylgeranyl diphosphate to the taxadiene precursor of taxol catalyzed by recombinant taxadiene synthase

Background: The committed step in the biosynthesis of the anticancer drug t axol in yew (Taxus) species is the cyclization of geranylgeranyl diphosphat e to taxa-4(5),11(12)-diene. The enzyme taxadiene synthase catalyzes this c omplex olefin cation cyclization cascade involving the formation of three r ings and three stereogenic centers. Results: Recombinant taxadiene synthase was incubated with specifically deu terated substrates, and the mechanism of cyclization was probed using MS an d NMR analyses of the products to define the crucial hydrogen migration and terminating deprotonation steps. The electrophilic cyclization involves th e ionization of the diphosphate with closure of the A-ring, followed by a u nique intramolecular transfer of the C11 proton to the reface of C7 to prom ote closure of the B/C-ring juncture, and cascade termination by proton eli mination from the beta -face of C5. Conclusions: These findings provide insight into the molecular architecture of the first dedicated step of taxol biosynthesis that creates the taxane carbon skeleton, and they have broad implications for the general mechanist ic capability of the large family of terpenoid cyclization enzymes.