Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi
Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi
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DOI：
10.1073/pnas.0608426104
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发表时间：
2007-02-27
影响因子：
11.1
通讯作者：
Sassa, Takeshi
中科院分区：
文献类型：
--
作者：
Toyomasu, Tomonobu;Tsukahara, Mai;Sassa, Takeshi
褐孢菌素是由植物病原真菌杏仁拟南星产生的一类二萜糖苷类化合物。褐球蛋白作为14-3-3蛋白的调节剂，在植物中是质膜H+-ATPase的强效激活剂，在动物细胞中也具有独特的生物活性。尽管它们的生物活性得到了很好的研究，但还没有发现编码褐霉素生物合成酶的基因。环状二萜通常是通过C-20前体香叶基香叶基二磷酸(GGDP)的环化反应合成的，GGDP是由通用的C-5异戊二烯单元二甲基烯丙基二磷酸和异戊烯基二磷酸通过戊基转移酶缩合而成的。我们发现，(+)-fusicocca-2，10(14)-diene是一种由C-5异戊二烯单元生物合成的三环烃前体，它是由一种特殊的多功能酶--杏仁褐变二烯合成酶(PAFS)生物合成的，该酶同时显示了戊烯转移酶和萜烯环合酶的活性。截短突变体和定点突变的功能分析表明，PAF由两个结构域组成：N端有一个萜烯环化酶结构域，C端有一个戊烯转移酶结构域。这些发现表明，无论GGDP的可用性如何，通过使用C5前体，可以在真菌中高效地产生镰刀菌烯。事实上，PAF的异源表达导致了2，10(14)-二烯在Escherichia cofi细胞中的积累，而在表达赤霉菌Ent-Kaurene合成酶的大肠杆菌细胞中没有检测到产物，Gibberella fujikuroi ent-Kaurene合成酶是另一种真菌二萜环化酶，也使用GGDP作为底物，但不包含异戊烯转移酶结构域。基因组行走表明，褐霉素类生物合成酶在PAFS基因附近编码为一个基因簇。
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, A 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 C5 precursors, irrespective of GGDP availability. In fact, heterologous expression of PaFS alone resulted in the accumulation of fusicocca 2,10 (14)-diene in Escherichia cofi 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.