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標題: X-ray繞射技術解析植物病原菌Xanthomonas campestris具重要代謝功能蛋白的結構研究
Structural studies of proteins with important metabolic functions from a plant pathogen Xanthomonas campestris using X-ray diffraction
作者: 蔡穎德
Tsai, Ying-Der
關鍵字: metabolic;代謝
出版社: 生物化學研究所
Xanthomonas campestris pv. campestris (X. campestris)屬於葛蘭氏陰性菌,為兼具學術性及應用性之菌種。X. campestris為感染十字花科造成十字花科黑腐病之植物病原菌,屬世界性之病害。Xanthomonas campestris pv. campestris str. ATCC 33913之基因體定序已由巴西團隊完成且發表;本土菌株Xanthomonas campestris pv. campestris str. 17也由台灣團隊完成定序及基因註解。結構基因體學最終目標為解析整個基因體蛋白質之結構,同時希望以結構生物學的角度研究蛋白分子層次功能,與其他蛋白之間的交互作用。本研究挑選了X. campestris str. 17菌株16個蛋白質,其中XC1236、XC1258和XC1936為適合X-ray繞射法研究結構的蛋白質。XC1236為447個胺基酸組成,由生物資訊的方法得知為一乙醯穀胺酸(acetylglutamate kinase)激酶,其為磷酸化乙醯穀胺酸(N-acetyl-L-glutamate)。其重要性在於此為合成精胺酸(arginine) 不可或缺的反應。精胺酸為生物體內必需的化合物,除了係合成蛋白的原料外,更為生成尿素的中間物,參與如ornithine代謝反應的前驅物。XC1236目前致力於改善其晶體的大小及提高其繞射數據分辨率(resolution)。XC1936為240個胺基酸組成,由生物資訊的方法得知為一尿苷酸激酶。其機制為催化ATP的γ-磷酸轉移給UMP,形成UDP。由於XC1936與同功能的已知結構蛋白序列相似度相當高(55%),故我們嘗試以分子取代 (molecular replacement)法來解析其三級結構。XC1258由266個胺基酸組成, XC1258 在不同原核生物體皆有相似序列,由活性區域的保留序列推測為一具碳氮水解酶功能的蛋白。碳氮水解酶廣泛的參與非胜肽鏈的水解作用,生成一些重要的天然物,如biotin、auxin和一些抗生素的前驅物。但目前僅僅由比對序列得知其含一由Glu-Lys-Cys所組成的未知功能催化區,尚無足夠的結構證據來證明這個催化反應。於本研究我們得到XC1258有Selenomethionine標定的結晶(約1.75 Å),已進行多波長異常繞射數據收集,並得到初步的三級結構。接下來將繼續進行結構的建立與修正,以得到最完整的XC1258的三級結構。

Xanthomonas campestris pv. campestris is a gram-negative bacterium and an important pathovar both academically and industrially. X. campestris is a bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. The genome of Xanthomonas campestris pv. campestris str. ATCC 33913 was sequenced by ONSA/FAPBSP/Brazil group in 2002 and by an integrated structural and functional group in Taiwan in 2002. The goal of the structural genomics is to determine the three-dimensional structures of all proteins on a genome wide scale and to study the protein function from the perspective of structure biology at the same time. In this thesis we aim to identify and characterize the three-dimensional structures of several proteins in the X. campestris using X-ray crystallography. In the respect, three proteins, XC1236, XC1258, and XC1936, were found to be suitable for structural studies by X-ray crystallography from screening of 16 target proteins. XC1236 is a 447 amino acids protein that was annotated as an acetylglutamate kinase by a bioinformatics approach, which phosphorylates the N-acetyl-L-glutamate. This reaction is essential for the synthesis of arginine, which is an essential component of all living beings, a precursor for energy storage, and an intermediate in the production of urea and ornithine. We try to improve XC1236 crystal size and resolution. XC1936 is a 240 amino acids protein, which was annotated as a uridylate kinase by a bioinformatics approach. Its main function is to catalyze phosphate transfer from theγposition of ATP to UMP to form UDP. We try to use molecular replacement method to solve XC1936 structure. XC1258 is a 266 amino acids protein, and many sequences with high similarity with XC1258 were found in different prokaryotes. It was annotated as a CN-hydrolase from the conserved activity site sequence. The CN-hydrolase superfamily proteins are involved in a wide variety of non-peptide carbon-nitrogen hydrolysis reactions, producing some important natural products such as auxin, biotin, precursors of antibiotics etc. We have obtained XC1258 Se-labeled crystals in a variety of forms suitable for X-ray diffraction studies (to at least 1.75 Å resolution). We have successfully applied the multiwavelength anomalous diffraction (MAD) method to determine its preliminary crystal structure. The structural refinement is currently udergoing.
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