Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi.
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.
Toyomasu
Tomonobu
T
Department of Bioresource Engineering, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan. toyomasu@tds1.tr.yamagata-u.ac.jp
Tsukahara
Mai
M
Kaneko
Akane
A
Niida
Rie
R
Mitsuhashi
Wataru
W
Dairi
Tohru
T
Kato
Nobuo
N
Sassa
Takeshi
T
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005658:Fungi / Q000737:chemistry* / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D006027:Glycosides / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009183:Mycotoxins / Q000096:biosynthesis*; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D006027:Glycosides; D009183:Mycotoxins; C007808:fusicoccin; D010453:Peptide Synthases