Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.
Structure and Function of Fusicoccadiene Synthase, a Hexameric Bifunctional Diterpene Synthase.
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DOI：
10.1021/acschembio.5b00960
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发表时间：
2016-04-15
影响因子：
4
通讯作者：
Christianson DW
中科院分区：
文献类型：
--
作者：
Chen M;Chou WK;Toyomasu T;Cane DE;Christianson DW
Fusicoccin A是由真菌病原体扁桃拟茎点霉（Phomopsis amygdali）产生的二萜葡糖苷植物毒素，该真菌病原体引起植物疾病缩窄溃疡，其在20世纪30年代首次在新泽西的桃园中发现。Fusicoccin A也是癌症化疗的一个新兴的新领导者。梭孢菌素A的烃前体是三环二萜梭孢菌素二烯，其由双功能萜类合酶产生。在这里，我们报告的X-射线晶体结构的fusiclidene合酶的各个催化域：C-末端结构域是一个链延伸酶，产生geranylgeranyl二磷酸，和N-末端结构域催化的geranylgeranyl二磷酸环化，形成fusiclidene。与双膦酸盐底物类似物复合的每个结构域的晶体结构表明，三个金属离子和三个带正电荷的氨基酸侧链在每个活性位点触发底物电离。虽然在体外孵育表明，环化酶结构域可以利用法呢基二磷酸和香叶基二磷酸作为替代底物，这些较短的类异戊二烯二磷酸主要转化为无环醇或烃产物。凝胶过滤色谱和分析超离心实验表明，全长fusiclidene合酶采用六聚体四级结构，和小角X-射线散射数据产生一个明确的分子包络，说明一个合理的模型六聚体组装。
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 1930’s. 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.