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標題: Xanthomonas campestris pv. campestris 未知功能蛋白之大量表達與結構分析
Cloning, expression and structural analysis of conserved and hypothetical proteins in the plant pathogen Xanthomonas campestris pv. campestris
作者: 楊超宇
Yang, Chao-Yu
關鍵字: hypothetical protein;未知功能蛋白;X-ray crystallography;nuclear magnetic resonance;X光晶體繞射;核磁共振
出版社: 生命科學系
Xanthomonas campestris pv. campestris屬於格蘭氏陰性菌。為兼具學術性及應用性之菌種。Xanthomonas campestris pv. campestris能分泌多種胞外蛋白,為研究格蘭氏陰性菌蛋白分泌之模式系統。其轉錄單位多以單基因方式為之,不具cAMP receptor但以多元調控蛋白Clp (cAMP receptor protein-like protein)協助執行調控功能。Xanthomonas campestris pv. campestris為一株感染十字花科造成十字花科黑腐病之植物病原菌,易生長於高溫多濕之環境,屬世界性之病害。且Xanthomonas campestris pv. campestris所產生之多醣體Xanthan為重要之工業原料。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。
結構基因體學之目標為解析出整個基因體之蛋白質三級結構,同時希望以結構生物學的角度研究Xanthomonas campestris pv. campestris蛋白之功能。本研究利用X-ray晶體繞射與高磁場核磁共振儀(NMR)技術研究Xanthomonas campestris pv. campestris str. 17菌株之9個目標蛋白質,並得到兩個(XC 2714與XC 6422)適合結構分析的蛋白質。XC 2714由160個胺基酸組成,為一種bacterioferritin comigratory protein(BCP),利用生物資訊的方法比對Pfam 與COG 資料庫後發現XC2714可被歸類為Thiol-specific antioxidant protein(TSA)/ Alkyl hydroperoxide peroxidase C(AhpC)family(pfam00578)或peroxiredoxin(COG1225)。直至目前為止,仍然沒有任何bacterioferritin comigratory protein(BCP)蛋白之三級結構被解析出來。因此我們利用收集的2D 15N-1H HSQC、3D HNCACB、3D CACB (CO)NH、HNCO、3D H(CC-CO)NH-TOCSY、3D (H)C(C-CO)NH-TOCSY等NMR光譜,完成大部分1H、15N以及13C之共振頻率判讀。以期提供此蛋白往後在結構上或者是功能上特徵的一個依據。XC 6422則由220個胺基酸組成,為未知功能蛋白。利用在Escherichia coli中大量表現此蛋白,並且經過結晶與X-ray晶體繞射實驗後,利用Se-MAD方式目前已成功的獲得XC 6422之三級結構。觀察其三級結構發現XC 6422具有Ser-Asp-His catalytic triad與Gly-X-Ser-X-Gly 序列。由於此為serine蛋白酶在催化上的重要特徵,所以XC 6422很有可能是以serine蛋白酶的機制水解蛋白質。

Xanthomonas campestris pv. campestris is a gram-negative bacterium and an important pathovar both academically and industrially. Xanthomonas campestris pv. campestris can secret many kinds of extracellular proteins, and is a model system for studying protein secretion of gram-negative bacteria. Although it doesn’t have cAMP-receptor, it carry out regulatory function by using Clp(cAMP-receptor protein-like protein), a multiple regulatory protein. Xanthomonas campestris pv. campestris is a bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. However, it also produces exopolysaccharide (xanthan) that is of great industrial importance. 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 of the Xanthomonas campestris pv. campestris 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 Xanthomonas campestris pv. campestris using X-ray crystallography and high-resolution NMR techniques. In this respect, two proteins, XC 2714 and XC 6422, are found to be suitable for structural studies from screening of 9 target proteins by X-ray crystallography and NMR analysis. XC 2714 is a putative bacterioferritin comigratory protein that contains 160 amino acids. It is classified either as a member of AhpC/TSA (pfam00578) in the Pfam database or as a member of peroxiredoxin (COG1225) in the COG database based on a bioinformatics approach. Until now, no tertiary structure of the bacterioferritin comigratory protein(BCP)has been determined. We have finished the nearly complete resonances for all the 1H, 15N, 13C nuclei of XC 2714 using 2D 15N -1H HSQC and triple resonance experiments including 3D- HNCACB、3D-CACB(CO)NH、HNCO、3D-H(CC-CO)NH-TOCSY、3D-(H)C(C-CO)-NH-TOCSY etc. These assignments can serve as a basis for further structural and functional characterization of this novel peroxiredoxins. XC 6422 from the plant pathogen Xanthomonas campestris pv. campestris is a hypothetical protein. A BLASTp search using this sequence revealed that most of the homologous sequences are annotated as hypothetical proteins. XC 6422, with a molecular mass of 23.98 kDa, has been overexpressed in Escherichia coli, purified, crystallized. The crystals were diffracted to good resolution. We have solved the final structure of XC 6422 using multiwavelength anomalous diffraction (Se-MAD) method. The structure of XC 6422 includes two features known to be important for serine protease catalysis, such as the Ser-Asp-His catalytic triad and Gly-X-Ser-X-Gly sequence motif. The structural features of XC 6422 suggest a serine protease mechanism for protein degradation.
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