Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase

Abstract

Fusicoccin A is a diterpene glucoside phytotoxin generated by the fungal pathogen Phomopsis amygdali that causes the plant disease constriction canker, first discovered in New Jersey peach orchards in the 1930s. Fusicoccin A is also an emerging new lead in cancer chemotherapy. The hydrocarbon precursor of fusicoccin A is the tricyclic diterpene fusicoccadiene, which is generated by a bifunctional terpenoid synthase. Here, we report X-ray crystal structures of the individual catalytic domains of fusicoccadiene synthase: the C-terminal domain is a chain elongation enzyme that generates geranylgeranyl diphosphate, and the N-terminal domain catalyzes the cyclization of geranylgeranyl diphosphate to form fusicoccadiene. Crystal structures of each domain complexed with bisphosphonate substrate analogues suggest that three metal ions and three positively charged amino acid side chains trigger substrate ionization in each active site. While in vitro incubations reveal that the cyclase domain can utilize farnesyl diphosphate and geranyl diphosphate as surrogate substrates, these shorter isoprenoid diphosphates are mainly converted into acyclic alcohol or hydrocarbon products. Gel filtration chromatography and analytical ultracentrifugation experiments indicate that full-length fusicoccadiene synthase adopts hexameric quaternary structure, and small-angle X-ray scattering data yield a well-defined molecular envelope illustrating a plausible model for hexamer assembly.

Figure 1. Fusicoccadiene synthase from P. amygdali (PaFS)

Geranylgeranyl diphosphate (GGPP) is generated from one molecule of dimethylallyl diphosphate (DMAPP) and three molecules of isopentenyl diphosphate (IPP) in the C-terminal α domain (blue box), and GGPP is cyclized in the N-terminal α domain to form fusicocca-2,10(14)-diene (red box). Further biosynthetic modifications (green) yield the phytotoxin fusicoccin A.

Figure 2. Catalytic activity measurements

(a) Generation of hydrocarbon products from substrates GPP, FPP, and GGPP in the absence of IPP (the concentrations of full-length PaFS in these experiments were 74.5 nM, 7.45 nM, and 14.9 nM, respectively). Catalysis with GPP exhibits Michaelis-Menten kinetics, whereas catalysis with FPP or GGPP exhibits cooperativity based on the sigmoidal dependence of catalytic activity on substrate concentration. With GGPP, substrate inhibition is evident at higher concentrations. (b) PaFS₃₅₀ (33.5 nM) exhibits Michaelis-Menten kinetics for the generation of hydrocarbon products from substrates GPP, FPP, and GGPP. (c) Proposed cyclization mechanism for the generation of fusicoccadiene (black and blue arrows) and δ-araneosene (black and red arrows) by full-length PaFS and PaFS₃₅₀ (a third, unidentified diterpene product is also generated).

Figure 3. PaFS C-terminal GGPP synthase domain

a) PaFS_CT adopts the α fold of a class I terpenoid synthase and crystallizes as a hexamer, or a trimer of dimers. The N-termini of selected subunits are labeled and indicate the point of connection to the missing N-terminal domain. Two perpendicular orientations are shown. (b) The binding of 3 Co²⁺ ions and pamidronate triggers complete closure of the active site of PaFS_CT. (c) Stereoview of intermolecular interactions in the PaFS_CT-Co²⁺₃-pamidronate complex. Metal coordination and hydrogen bond interactions are shown as black and red dashed lines, respectively (metal-ligand distances are recorded in Supplementary Information Table S3). Pamidronate binds in the DMAPP binding site, so interactions of the phosphonate groups mimic interactions with the diphosphate group of DMAPP that trigger ionization and formation of the allylic cation that initiates the chain elongation reaction.

Figure 4. PaFS N-terminal GGPP cyclase domain

(a) Stereoview of the PaFS₃₄₄-Mg²⁺₃-pamidronate complex reveals that the N-terminal domain adopts the α fold of a class I terpenoid synthase. (b) Stereoview of the PaFS₃₄₄-Mg²⁺₃-pamidronate complex showing metal coordination and hydrogen bond interactions as black and red dashed lines, respectively (metal-ligand distances are recorded in Supplementary Information Table S3). (c) The G60-F65 hairpin segment may facilitate communication between cyclization domain active sites in full-length hexameric PaFS by mediating interactions between active site residue F65 and interdomain linker residues N333 and Q336 (green).

Figure 5. Models of enzyme-product complexes

(a) Model of product fusicoccadiene bound in the active site of PaFS₃₄₄. The three-dimensional active site contour is represented by black meshwork, and fusicoccadiene was manually fit into this meshwork. The position of the Mg²⁺₃-PP_i cluster is modeled after the Mg²⁺₃-bisphosphonate cluster in the PaFS₃₄₄-Mg²⁺₃-pamidronate complex. Two perpendicular views are shown; the backbone carbonyl of V192 at the helix G break (δ⁻) is oriented toward the location of a proposed carbocation intermediate in the PaFS mechanism. (b) Cut-away view of the active site pocket of PaFS₃₄₄, showing how the surface contour (black meshwork) is defined by residues lining the pocket. (c) Ophiobolin F docked in the active site of ophiobolin F synthase modeled after the PaFS₃₄₄-fusicoccadiene complex in (b). The extra active site volume resulting from the W225L and V228A substitutions readily accommodate the larger C₂₅ sesterterpene.

Figure 6. Structure of the full-length PaFS hexamer in solution

(a) Model of the hexamer of full-length N333A/Q336A PaFS fit into the three-fold averaged ab initio molecular envelope generated from SAXS data. PaFS₃₄₄ and PaFS_CT dimers are marine and green, respectively. (b) Theoretical scattering profile (teal solid line) overlaid with experimental data (black) indicates an excellent fit of the molecular envelope in (a), with χ = 1.5 as calculated with SASREF.