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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林俊宏(Chun-Hung Lin) | |
| dc.contributor.author | Hung-Yu Chiang | en |
| dc.contributor.author | 江宏宇 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:43:22Z | - |
| dc.date.copyright | 2016-08-25 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-16 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19048 | - |
| dc.description.abstract | 岩藻糖 (fucose) 常修飾在寡醣鏈末端，在許多生理作用上扮演不可或缺的重要性。鬆脆桿菌 (Bacteroides fragilis) 為人類腸道常見的共生菌之一；鬆脆桿菌會由宿主腸胃道表皮細胞上之末端取得岩藻糖，並利用岩藻糖生合成的補救途徑 (salvage pathway) 來合成GDP-L-fucose，以提高表面路易士抗原的表現量，達到躲避宿主的免疫作用。與其他物種不同，鬆脆桿菌將L-fucokinase (FK) 及GDP-L-fucose pyrophosphorylase (GFPP) 合而為一，而具有雙功能之蛋白 (稱為FKP)。本研究以酵素動力學和蛋白質結構來探討這個雙功能蛋白的結構與功能。根據蛋白質序列比對，我們將全長FKP (FL-FKP)、以及含有單一酵素功能的FK (518-949)、GFPP (1-496) 在大腸桿菌上表現、生產、純化後達到 > 95% 的純度，它們酵素動力學分析指出， FL-FKP所含FK活性與FK518-949比較起來，前者在Km (fucose)上比後者低10倍，在kcat (fucose) 和kcat (ATP) 的測量上，全長較單一活性區域高2倍。以GFPP活性作比較，FL-FKP和GFPP1-496兩者在kcat (fucose-1-P; f1p) 及kcat (GTP) 上，前者均較後者高出兩倍。此外，以分析型超高速離心法分析FL-FKP在緩衝溶液中的四級結構大多為三聚體。在x-ray結晶解析上，為了解決GFPP相位問題，我們將SUMO蛋白接至GFPP蛋白N端上並表現得到GFPP單體，並成功解析出它的結構 (解析度2.35 Å)，最後藉由GFPP、FK結構解析出FL-FKP 之結構 (解析度2.37 Å)。我們藉由結構、與其他相似蛋白結構比較及定點突變方式，發現FK活性區段催化是以D762為general base，作用在fucose C-1的羥基上；W599、D601及Q761負責與fucose的結合作用；R592、E751、S719及S720則與ATP的結合有關。這些資訊讓我們提出FK可能催化機制。除此之外，我們嘗試將兩個酵素活性之間的連結區域 (序列431-583) 作部分剔除，並無法表現可溶性之蛋白，發現這個區域提供活性與結構穩定的必要性。 | zh_TW |
| dc.description.abstract | Fucosylation is one of the most common modifications of oligosaccharides on glycoproteins and glycolipids. These fucosylated glycans usually function as special epitopes that contribute to specific physiological effects, including host-microbe interactions. Bacteroides fragilis is known to be one of the most common commensal bacteria in the human gastrointestinal tract. To avoid the host’s immune system, B. fragilis uses salvage pathways to utilize fucose from the host to synthesize fucosylated Lewis antigens, which these bacteria then express on the cell surface. In contrast to other species, B. fragilis combines L-fucokinase and GDP-L-fucose pyrophosphorylase into one bifunctional enzyme (called FKP). In this thesis, we studied the function and structure of FKP by enzyme kinetics and x-ray crystallography. The fragments corresponding to the FK domain (518-949 a.a.), the GFPP domain (1-430 a.a.) and the full-length FKP (FL-FKP) were successfully expressed in the E. coli expression system and isolated with greater than 95% purity. The resulting activity assay indicated that Km (fucose) from FL-FKP was ten-fold lower than that from FK584-949 and that kcat (fucose) and kcat (ATP) from FL-FKP were two-fold higher than that from FK. The GFPP activity results showed that kcat (fucose-1-P; f1p) and kcat (GTP) from FL-FKP were 2-fold higher than that from GFPP1-496. According to analytical ultracentrifugation (AUC) analysis, the quaternary structure of FKP was mostly trimeric. Using x-ray crystallography, we solved the phase problem of the structure of GFPP by fusing a SUMO protein to the N terminus of GFPP and then elucidated its structure (resolution 2.35 Å). Finally, we determined the structure of FL-FKP (resolution 2.37 Å) using the GFPP and FK domain structures. To ascertain the catalytic mechanisms of FK, we used the structure, superimposed structure and site-directed mutagenesis, and proposed that D762 acts as a general base for interacting with the fucose C-1 OH group, W599, D601, and Q761 are responsible for binding to fucose, and R592, E751, S719, and S720 are involved with binding to ATP. Moreover, protein expressions of the partially truncated linker of FKP (431-583 a.a.) are found to result in inclusion bodies, showing that this region is essential for structural intrgrity or/and stability. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:43:22Z (GMT). No. of bitstreams: 1 ntu-105-R03b46033-1.pdf: 15533904 bytes, checksum: 6d72dd2e8c4f847745281571d8d998c5 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 口試委員會審定書 ...#
致謝 ...i 中文摘要 ...iii ABSTRACT ...iv 目錄 ...v 圖目錄 ...ix 表目錄 ...xi Chapter 1 緒論 ...1 1-1 人類腸道生態 (The Human Intestine Ecosystem) ...1 1-2 擬桿菌屬 (Bacteroides) 與人類之關係 ...1 1-2-1 擬桿菌屬 (Bacteroides) ...1 1-2-2 鬆脆桿菌 (Bacteroides fragilis) 與岩藻糖之關聯 ...2 1-3 岩藻糖及其生物功能 ...3 1-4 岩藻糖基化醣鏈之生合成及代謝相關酵素 ...3 1-5 ABO血型抗原與路易士抗原之角色 ...5 1-5-1 ABO血型抗原 (ABO Blood Group Antigen) ...5 1-5-2 路易士抗原 (Lewis Antigen) ...5 1-6 鬆脆桿菌之雙功能岩藻糖激酶/鳥苷二磷酸岩藻糖焦磷酸化酶及應用 ...7 1-6-1 補救途徑中之鳥苷二磷酸岩藻糖合成酶 (GDP-fucose synthetase) ...7 1-6-2 鬆脆桿菌之雙功能岩藻糖激酶/鳥苷二磷酸岩藻糖焦磷酸化酶 (L-fucokinase/GDP-L-fucose pyrophosphorylase) ...7 1-6-3 鬆脆桿菌FKP之晶體討論 ...9 1-6-4 鬆脆桿菌FKP之生化應用 ...9 1-7 論文研究動機 ...11 Chapter 2 材料與方法 ...12 2-1 FKP重組蛋白之基因轉殖 ...12 2-1-1 FKP之基因取得 ...12 2-1-2 引子設計 ...12 2-1-3 FKP之linker truncated 引子設計 ...13 2-1-4 聚合酶連鎖反應 ...13 2-1-5 黏合反應 ...14 2-1-6 融合蛋白之質體建構 ...15 2-1-7 定點突變 ...16 2-1-8 轉殖作用 ...16 2-1-9 質體DNA之抽取 ...17 2-2 FKP重組蛋白之表現及純化 ...17 2-2-1 重組蛋白之誘導與大量表現 ...17 2-2-2 硒代蛋氨酸標定蛋白之誘導與大量表現 ...17 2-2-3 重組蛋白之純化 ...18 2-2-4 蛋白質定量 ...18 2-3 FKP 之特性及活性鑑定 ...19 2-3-1 偵測FKP活性區間催化之最適pH值 ...19 2-3-2 偵測FKP活性區間催化之最適金屬離子 ...21 2-3-3 酵素動力學參數計算 ...22 2-3-4 岩藻糖激酶FK酵素動力學參數偵測 ...22 2-3-5 鳥苷二磷酸岩藻糖焦磷酸化酶GFPP酵素動力學參數偵測 ...24 2-3-6 分析型超高速離心 (Analysis Ultracentrifuge; AUC ) ...25 2-3-7 粒徑篩層析-多角度光散射分析系統 (Size Exclusion Column- Multi-angle Light Scattering; SEC-MALS) ...25 2-4 FKP之X光晶體學 ...26 2-4-1 養晶條件之篩選 (Screening) 與觀察 ...26 2-4-2 養晶條件之優化及培養 ...26 2-4-3 X光繞射實驗及數據收集與處理 ...26 2-4-4 解決相位角問題 ...27 2-4-5 結構之建立及精緻化 ...27 2-4-6 蛋白質結構圖形之作圖 ...27 Chapter 3 結果與討論 ...28 3-1 FKP與其片段區間重組蛋白之表現及純化 ...28 3-1-1 序列比對 ...28 3-1-2 質體建構 ...29 3-1-3 蛋白質表現 ...29 3-1-4 蛋白質純化 ...30 3-2 FKP重組蛋白之活性及生化特性分析 ...30 3-2-1 pH值對FKP之活性影響 ...30 3-2-2 二價金屬離子對於FKP之活性影響 ...31 3-2-3 岩藻糖激酶 (FK) 之酵素動力學參數偵測 ...32 3-2-4 鳥苷二磷酸岩藻糖焦磷酸化酶 (GFPP) 之酵素動力學參數偵測 ...33 3-2-5 FKP之linker重要性 ...33 3-2-6 FKP與其片段之四級結構探討 ...34 3-3 FKP之蛋白質結構 ...34 3-3-1 FL-FKP、GFPP1-496蛋白質晶體之培養 ...34 3-3-2 SUMO-GFPP1-496蛋白質晶體之培養 ...35 3-3-3 結晶學數據 ...35 3-3-4 鬆脆桿菌FKP之整體結構 ...36 3-3-5 鬆脆桿菌FKP之岩藻糖激酶整體結構 ...36 3-3-6 岩藻糖激酶與受質之交互作用 ...37 3-3-7 岩藻糖激酶之反應機制探討 ...37 3-3-8 岩藻糖激酶之定點突變 ...38 3-3-9 鬆脆桿菌FKP之鳥苷二磷酸岩藻糖焦磷酸化酶 (GFPP) 整體結構 ...38 3-4 討論 ...39 3-4-1 鬆脆桿菌FKP包含兩區段之優勢 ...39 3-4-2 FKP之其區段蛋白之穩定性 ...40 3-4-3 FKP兩區段蛋白間Linker之探討 ...40 3-4-4 FKP之四級結構之討論 ...41 3-4-5 FKP結構之應用 ...41 Chapter 4 結論 ...42 圖 ...44 表 ...64 參考資料 ...69 | |
| dc.language.iso | zh-TW | |
| dc.subject | 鬆脆桿菌 | zh_TW |
| dc.subject | 岩藻糖 | zh_TW |
| dc.subject | x光結晶學 | zh_TW |
| dc.subject | 酵素動力學 | zh_TW |
| dc.subject | 鳥?二磷酸合成? | zh_TW |
| dc.subject | GDP-fucose synthetase | en |
| dc.subject | X-ray crystallography | en |
| dc.subject | L-fucose | en |
| dc.title | 鬆脆桿菌鳥苷二磷酸岩藻糖合成酶之結構分析 | zh_TW |
| dc.title | Structural analysis of Bacteroides fragilis GDP-fucose synthetase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張崇毅(Chung-I Chang),何孟樵(Meng-Chiao Ho) | |
| dc.subject.keyword | 岩藻糖,鳥?二磷酸合成?,鬆脆桿菌,酵素動力學,x光結晶學, | zh_TW |
| dc.subject.keyword | L-fucose,GDP-fucose synthetase,X-ray crystallography, | en |
| dc.relation.page | 75 | |
| dc.identifier.doi | 10.6342/NTU201602732 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2016-08-17 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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