Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi

Abstract

Fusicoccins are a class of diterpene glucosides produced by the plant-pathogenic fungus Phomopsis amygdali. As modulators of 14-3-3 proteins, fusicoccins function as potent activators of plasma membrane H(+)-ATPase in plants and also exhibit unique biological activity in animal cells. Despite their well studied biological activities, no genes encoding fusicoccin biosynthetic enzymes have been identified. Cyclic diterpenes are commonly synthesized via cyclization of a C(20) precursor, geranylgeranyl diphosphate (GGDP), which is produced through condensation of the universal C(5) isoprene units dimethylallyl diphosphate and isopentenyl diphosphate by prenyltransferases. We found that (+)-fusicocca-2,10 (14)-diene, a tricyclic hydrocarbon precursor for fusicoccins, is biosynthesized from the C(5) isoprene units by an unusual multifunctional enzyme, P. amygdali fusicoccadiene synthase (PaFS), which shows both prenyltransferase and terpene cyclase activities. The functional analysis of truncated mutants and site-directed mutagenesis demonstrated that PaFS consists of two domains: a terpene cyclase domain at the N terminus and a prenyltransferase domain at the C terminus. These findings suggest that fusicoccadiene can be produced efficiently in the fungus by using the C(5) precursors, irrespective of GGDP availability. In fact, heterologous expression of PaFS alone resulted in the accumulation of fusicocca-2,10 (14)-diene in Escherichia coli cells, whereas no product was detected in E. coli cells expressing Gibberella fujikuroi ent-kaurene synthase, another fungal diterpene cyclase that also uses GGDP as a substrate but does not contain a prenyltransferase domain. Genome walking suggested that fusicoccin biosynthetic enzymes are encoded as a gene cluster near the PaFS gene.

Fig. 1.

Proposed biosynthetic pathway of fusicoccin A and J.

Fig. 2.

Chromosome map around PaFS and the primary structure of PaFS. (A) Chromosome map. A filled reverse triangle indicates the starting point of genome walking. The restriction sites for ScaI, DraI, EcoRV, FspI, PvuII, and StuI used in genome walking are represented by the letters C, D, E, F, P, and S, respectively. The direction and deduced region of transcription are represented by arrows. The deduced orf1, orf2, orf3, orf4, and orf5 represent 2-oxo-glutarate-dependent dioxygenase, P450 monooxygenase, short-chain dehydrogenase/reductase, α-mannosidase, and Nop14 like genes, respectively. Homology searches were performed by using BLAST (www.ncbi.nlm.nih.gov/BLAST/). (B) Schematic of two putative domains of PaFS. Black and gray bars indicate the terpene cyclase domain and the prenyltransferase domain, respectively. Reverse open triangles indicate the DDxxD motives of both domains. The borders between the terpene cyclase domain and the prenyltransferase domain were deduced by the homology with other fungal aristolochene synthases or GGSs.

Fig. 3.

GC–MS results for products converted from GGDP by GST-PaFS. (A) Total ion chromatograms of the products after incubation of GGDP with the recombinant GST-PaFS. (B) Total ion chromatogram of the hydrocarbon fraction obtained from mycelia of P. amygdali N2. (C) Full-scan mass spectrum of peak 1. (D) Full-scan mass spectrum of authentic fusicocca-2,10 (14)-diene (peak 2).

Fig. 4.

Cyclase and prenyltransferase activity of mutant proteins. Black and gray bars indicate the putative terpene cyclase domain and the prenyltransferase domain, respectively. Reverse open triangles indicate the aspartate-rich motif in each domain. CA, cyclase activity converting GGDP into fusicocca-2,10 (14)-diene; PA, prenyltransferase activity converting FDP and IPP into GGDP. (A) Truncated mutants. The calculated molecular mass of N334, N390, N436, N493, and C385 are 38, 44, 49, 56, and 43 kDa, respectively. The wild-type PaFS is a 719-aa polypeptide, and its molecular mass is 81 kDa. (B) Site-directed mutagenesis study. The aspartate residue in each domain was substituted by an alanine residue. (+), detected; (−), not detected. The activities of the mutant enzymes shown as (+) were almost the same as those of the wild type.

Fig. 5.

Accumulation of fusicocca-2,10 (14)-diene in E. coli cells carrying GST-PaFS. (A) CBB-stained SDS/PAGE gel to check the recombinant protein expression: lane 1, GST (vector control); lane 2, GST-GfCPS/KS (ent-kaurene synthase from G. fujikuroi, ≈130 kDa); lane 3, GST-PaFS (≈110 kDa). Open triangles show each recombinant protein. (B) Total ion chromatograms of the hydrocarbon fraction obtained from E. coli cells expressing GST-PaFS. The mass spectrum of peak 3 was identical to that of both peaks 1 and 2.

Fig. 6.

Synthesis of fusicocca-2,10 (14)-diene from isoprene units by PaFS. This enzyme is a diterpene hydrocarbon synthase possessing both prenyltransferase and terpene cyclase activity.