Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92200
標題: 從結構探討環鳥糞嘌呤核苷酸及環雙鳥糞嘌呤核苷酸控制的細菌訊息傳導機制
The structral aspects of cyclic GMP and cyclic di-GMP-dependent bacterial signaling pathways
作者: 楊詔貴
Jauo-Guey Yang
關鍵字: 環鳥糞嘌呤核苷酸;環雙鳥糞嘌呤核苷酸;二元傳遞訊息系統;XC_0249;XC_0250;RpfG;cyclic GMP;cyclic di-GMP;two component signal transduction;XC_0249;XC_0250;RpfG
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摘要: 
二級訊息傳導物質Cyclic guanosine 3',5'-monophosphate (cyclic GMP) 在細菌訊號傳遞中所扮演的功能在過去並非相當明確。然而在植物病原菌Xanthomonas campestris pv campestris (Xcc)的基因篩選實驗中發現,編碼XC_0250會轉譯出第三型nucleotidyl 環化酶,並合成cyclic GMP。在in vitro的實驗也證實純化出來的XC_0250具有合成cyclic GMP的能力。而其相連的基因XC_0249,被發現是一個帶有cyclic mononucleotide-binding (cNMP) domain以及 GGDEF diguanylate cyclase domain的蛋白。實驗發現,在cyclic GMP存在的條件下會提升XC_0249的cyclic di-GMP 合成能力。本篇研究我們解析了XC_0249 cNMP domain和cyclic GMP的複合體結構,並從結構的角度探討cyclic GMP和cyclic di-GMP是如何互相影響進而調控下游的生物生理機轉。而突變XC_0250或XC_0249的Xcc都將降低對植物的毒性以及降低生物膜的生合成。這樣的發現也因此連接了cGMP藉由影響cyclic di-GMP的合成調控整個訊息傳導路徑。除此之外,我們也嘗試著解析會和XC_0249的同源蛋白SM_0233交互作用的蛋白SM_RpfGD81E以及SM_RpfGD81E和SM_0233 GGDEF domain以及其cyclic di-GMP binding form的複合體結構,目前已初步得到SM_RpfGD81E-cyclic di-GMP的粗略結構。

Cyclic guanosine 3',5'-monophosphate (cyclic GMP) is a second messenger whose role in bacterial signaling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified XC_0250 as a protein with a class III nucleotidyl cyclase domain, which is required for cyclic GMP synthesis. Purified XC_0250 is active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide-binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di-GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 contains a cyclic GMP binding site and a structure–function analysis, directed by determination of the crystal structure of the holo-complex and enzymatic assay confirm the cyclic GMP binding residues, demonstrated the site of cyclic GMP binding that modulates cyclic di-GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector XC_0249 that directly links cyclic GMP and cyclic di-GMP signaling. Besides, we also tried to determine the structure of SM_RpfGD81E which can interact with GGDEF domain of SM_0233, a XC_0249 homologous protein. We have obtained the crude full length SM_RpfGD81E crystal structure in complex with cyclic di-GMP. Further refinement is necessary to truly reveal its c-di-GMP binding mode.
URI: http://hdl.handle.net/11455/92200
Rights: 同意授權瀏覽/列印電子全文服務,2015-01-22起公開。
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