Terpenoid synthase reaction sequences are among the most complex carbon-carbon bond forming reactions in nature, since approximately two-thirds of the carbon atoms in the substrates undergo changes in bonding and/or hybridization during a single, precisely controlled reaction sequence. Uniquely, fusicoccadiene synthase from Phomopsis amygdali (PaFS) catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions, blocking proliferation and migration of glioblastoma cells.<br/>PaFS establishes the structure and stereochemistry of the hydrocarbon skeleton of fusicoccin A. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Oligomerization of PaFS likely confers an “agglomeration effect,” in which substrate is channeled by proximity between active sites of consecutive reactions. Here, we describe full-length PaFS using cryo-electron microscopy, revealing a central octameric core of prenyltransferase domains with eight pendant cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Chemical crosslinking experiments additionally characterize compact conformations that can be achieved, in which cyclase domains are more closely associated with the prenyltransferase core.<br/>This structural analysis uncovers a framework for understanding substrate channeling between active sites, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene. The studies discussed herein will directly inform biosynthesis of pharmaceutically relevant terpenoids.

Structural insight on assembly-line catalysis in fusicoccadiene biosynthesis

ACS Fall 2021

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