Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23841
標題: 利用NMR及X-ray繞射技術研究Xanthomonas campestris表達的蛋白結構
Structural studies of proteins expressed by Xanthomonas campestris using NMR and X-ray techniques
作者: 郭威廷
Kuo, Wei-Ting
關鍵字: Xanthomonas campestris;十字花科黑腐病菌;NMR;X-ray crystallography;核磁共振;X光晶體繞射
出版社: 生物化學研究所
摘要: 
Xanthomonas campestris pv. campestris 屬於格蘭氏陰性菌,能分泌多種胞外蛋白,主要為感染十字花科,而造成十字花科黑腐病。此外 X. campestris 所產生之多醣體 Xanthan 也是重要之工業原料。 Xanthomonas 本土菌株 Xanthomonas campestris pv. campestris str. 17由台灣團隊完成定序及基因註解。它的ORF XC2258 由生物資訊的方法預測功能為一解酯酶 (esterase),在 Pfam 資料庫中屬於 α/β-hydrolase superfamily 家族。是普遍存在於生物界的一種酵素,但其確切功能尚未知。在序列比對中其最類似一種解酯酶 (esterase),其主要功能為打斷阿魏酸 (Ferulic acid) 間形成交聯的酯質鍵結 (ester bond ),進而造成植物軟化腐敗而死亡 (阿魏酸是細胞壁物質形成交聯的酯化酚類中的一種)。在微生物、植物及動物界,已有多種解酯酶 (esterase) 酵素被商業化生產,所以解酯酶是一種極重要且具應用潛力的生物觸媒, 而其中大部分是來自微生物(包括真菌和細菌)。此外利用另一預測軟體 GenTHREADER PREDICTION 預測也發現一個相似結構,其功能為酯化酵素蛋白 Acyl Protein Thioesterase。而這個酵素也具有相同的催化中心的高度保留胺基酸 Ser/His/Asp,利用一級序列的比對的所提供資訊有限仍不足解釋其正確的功能為何。為了更加了解 XC2258 其確切的功能及三級結構摺疊,已構築 XC2258 載體,並成功的表達及純化大量可溶蛋白,且篩選出其合適的結晶條件。 XC2258 的晶體經過 X-ray 繞射分析後其解析度可達 1.3 Å 。另外 XC4476 其預測功能分別為一胞嘧啶去氨酶 (cytosine deaminase ), 其為一催化胞嘧啶成為尿嘧啶過程中的去氨酵素, 並廣泛存在於原核生物及真菌中,但尚未於多細胞真核生物中發現。在哺乳動物細胞中,胞苷去氨酶 (cytidine deaminase) 則俱類似功能。此酵素亦可催化一個抗腫瘤藥物 5-Fluorouracil (5-FU) 及5-fluorocytosine (5-FC) 間的轉換。其中5-FU 是目前轉移性大腸、直腸癌化學治療最主要的藥物,其作用機轉主要是與 thymidylate synthase 結合並抑制其作用,導致 thymidylate 無法生成,進而影響DNA的合成。XC 5700 經由生物資訊的方法預測其功能為一轉譯調控因子蛋白 (transcriptional regulator protein)。XC5700 由66個胺基酸所組成,利用一級序列比對後發現。與一個腸球菌中一轉譯調控因子蛋白 CylR2 有高達 49% 序列相似度。 在腸球菌中的 CylR2 蛋白會與另一個蛋白 CylR1 組成一個雙成份控制系統 (two-component regulatory system) 並調控一個外毒素,溶細胞素 (cytolysin) 的分泌。目前我們已經利用非尋常散射法(anomalous dispersion method) 方法成功的解決 XC5700 相位角的問題。並已獲得一個XC5700初步結構。另外我們也試著以NMR解析XC60 、XC1128 、XC8、 XC5534 和 XC6732 等蛋白並已經製備同位素標定蛋白以供 NMR 實驗之需。

Xanthomonas campestris pv. campestris is a gram-negative bacterium and an important pathovar both academically and industrially. X. campestris can secret many kinds of extracellular proteins, and is a model system for studying protein secretion of gram-negative bacteria. X. 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 the Xanthomonas campestris pv. campestris str. ATCC 33913 was sequenced by a Brazil group and that of str.17 by an integrated structural and functional group in Taiwan in 2002. The XC 2258 ORF in X. campestris pv. campestris str. 17 was annotated as a esterase by a BLAST search. It cleaves the ester bond between arabinoxylan and pectin where the ferulic acid moieties cross-link the layers of polysaccharide chains within hemicellulose. The native XC2258 crystals were obtained from the purified recombinant protein and exhibits a variety of forms diffracted to at least 1.3 Å resolution by using X-ray crystallography. We have tried to solve the structure of XC2258 by the multiwavelength anomalous diffraction (MAD) method using selenomethionine-substituted protein. Now, we have obtained the selenomethionine-substituted protein crystals that exhibit a variety of forms diffracted to at least 3.0 Å resolution. The XC4476 in X. campestris pv. campestris str. 17 was annotated as a cytosine deaminase by a bioinformatics approach. Cytosine deaminase catalyzes the deamination of cytosine to uracil. The enzyme has been found in bacteria and fungi, but not in mammalian cells, which utilize cytidine deaminase (CDA) instead. Cytosine deaminase is an attractive drug target through its catalysis of the deamination of the prodrug 5-fluorocytosine to 5-fluorouracil. The XC5700 in X. campestris pv. campestris str. 17 was annotated as a transcriptional regulator protein
by a bioinformatics approach. It contains 66 amino acids and shares 49% identity with a protein from cytolysin regulator CylR2 from Enterococcus faecalis. Enterococcus faecalis is one of the major causes for hospital-acquired antibiotic-resistant infections. It produces an exotoxin, called cytolysin. The regulation of the cytolysin operon was connected to autoinduction by a quorum-sensing mechanism involving the CylR1/CylR2 two-component regulatory system. Now, we have successfully solve the XC5700 phase problem by the multiwavelength anomalous diffraction (MAD) method using selenomethionine-substituted protein and obtain an initial structure. Besides, we try to determine proteins structure in X. campestris pv by NMR. Until now we prepared isotope label proteins of XC60、XC1128、XC8、XC5534 and XC6732 and collected their NMR spectrum.
URI: http://hdl.handle.net/11455/23841
Appears in Collections:生物化學研究所

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