geranylgeranyl-pyrophosphate and fusicoccadiene

Fusicoccadiene synthase from the fungus

Topics: Alkyl and Aryl Transferases; Dimethylallyltranstransferase; Diterpenes

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.

Topics: Alkyl and Aryl Transferases; Ascomycota; Catalysis; Catalytic Domain; Cryoelectron Microscopy; Cyclization; Dimethylallyltranstransferase; Diterpenes; Fungal Proteins; Glycosides; Lyases; Multifunctional Enzymes; Polyisoprenyl Phosphates; Protein Conformation

Enzymatic cyclization of geranylgeranyl diphosphate to fusicoccadiene involves a transannular proton transfer process. Label distribution in the cyclized products derived from deuterium-labeled GGDPs showed that a proton generated from C-10 migrates to C-6 in the intermediary dolabellane framework prior to the second ring formation. Although a direct 1,5-proton transfer would achieve this process, semiempirical MO calculations suggested an alternative pathway, which involves successive 1,4- and 1,5-proton transfers using C-2 as a springboard.

Topics: Cyclization; Diterpenes; Models, Molecular; Molecular Structure; Polyisoprenyl Phosphates