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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王愛玉(Ai-Yu Wang) | |
| dc.contributor.author | Yu-Chiao Huang | en |
| dc.contributor.author | 黃玉嬌 | zh_TW |
| dc.date.accessioned | 2021-06-16T03:40:54Z | - |
| dc.date.available | 2020-03-16 | |
| dc.date.copyright | 2015-03-16 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-02-13 | |
| dc.identifier.citation | Absmanner, B., Schmeiser, V., Kampf, M. and Lehle, L. (2010) Biochemical characterization, membrane association and identification of amino acids essential for the function of Alg11 from Saccharomyces cerevisiae, an alpha1,2-mannosyltransferase catalysing two sequential glycosylation steps in the formation of the lipid-linked core oligosaccharide. Biochem J 426: 205-217.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54894 | - |
| dc.description.abstract | 蔗糖合成酶 (SuS) 催化蔗糖與 UDP 轉化為果糖與 UDP-glucose 的可逆反應。水稻中蔗糖合成酶 (RSuS) 至少由七種異構基因 (RSus) 所表現，且不同異構酶間的胺基酸序列高度相似。
本研究的目標是解析 RSuS 結構與功能間的關係。基質結合分析的結果顯示，RSuS 在蔗糖分解方向的先導基質為 UDP，而在蔗糖合成方向的先導基質為果糖。嘗試利用小角度 X-ray 散射、蛋白質結晶繞射及同源模擬的方式，進行 RSuS 蛋白質結構的解析。利用阿拉伯芥蔗糖合成酶 AtSuS1 為模版進行同源模擬，得到接近全長的 RSuS 模擬結構。由模擬結構的資訊推測，Gln307 及 E-X7-E motif 與基質結合相關。將 Gln307 突變成 Asn 或 Ser 會導致 RSuS1 酵素活性喪失，RSuS1(Q307N) 仍保有與果糖的結合能力，但 RSuS1(Q307S) 則喪失與果糖結合的能力。在糖基轉移酶 3、4 及 5 家族中，E-X7-E motif 是保守性高的區塊。此區塊中的突變株雖不影響 RSuS3 蛋白質四元體的形成，但僅 F680Y 與E686D 保有部分活性，E678D、E678Q、F680S 及 E686Q 則無酵素活性。對此區塊內無活性的突變株進行基質結合能力分析，結果顯示 E678D、E678Q、F680S 及 E686Q 皆保有與 UDP 結合的能力，但都無法與果糖結合。對 RSuS3 與保有活性的突變株 F680Y 及 E686D 進行酵素動力學分析，結果顯示這兩個胺基酸突變不僅影響酵素與 UDP-glucose 的結合能力，也影響酵素活性的展現。 | zh_TW |
| dc.description.abstract | Sucrose synthase (SuS, EC 2.4.1.13) catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. The enzyme is encoded by at least seven RSus genes in rice. The amino acid sequences of different RSuS are highly similar.
The objective of this study is to elucidate the structure-function relationships of RSuS. The results of substrate binding assay shown that UDP and fructose are the leading substrate of sucrose cleavage and sucrose synthesis direction, respectively. Different approaches including small angle X-ray scattering, protein crystallography and homology modeling, have been attempted to obtain the structure information of RSuS. The theoretical structure of partial RSuS, which was built with the structure of Arabidopsis sucrose synthase 1 as template, revealed that Gln307 and E-X7-E motif of RSuS was involved in fructose binding. Changing the Gln307 to Asn or Ser resulted in completely loss of enzyme activity. Substrate binding assays revealed that the mutant RSuS1(Q307N) retained the capability of fructose binding while RSuS1(Q307S) did not. The E-X7-E motif is conserved in members of glycosyltransferase family 4 (GT4) and other two GT families. The results of enzyme activity assay showed that the mutants E686D and F680Y retained partial enzyme activity and the mutants E678D, E678Q, F680S and E686Q were inactive; however, the tetrameric conformation was maintained in these mutant proteins. The results of substrate binding assays revealed that the mutants E6786D, E686Q, F680S and E686Q could bind UDP but lost the capability of sucrose binding and fructose binding. The kinetic parameters of RSuS3, F680Y and E686D indicated that the residues in E-X7-E motif are important in UDP-glucose binding and enzyme activity. | en |
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| dc.description.tableofcontents | 目錄 I
縮寫表 V 中文摘要 VII Abstract VIII 第一章 研究背景 9 第一節 蔗糖合成酶之生化性質 9 1.1 蔗糖合成酶之生化性質 9 1.2 蔗糖合成酶異構酶及異構基因 9 第二節 蔗糖合成酶之生理功能 10 2.1 參與蔗糖運輸 10 2.2 參與澱粉的生合成 11 2.3 參與細胞壁多醣類的生合成 12 第三節 蔗糖合成酶之細胞內定位及活性調控 13 3.1 蔗糖合成酶的膜結合形式與細胞內定位 13 3.2 蔗糖合成酶之磷酸化 13 3.3 蔗糖合成酶活性調控 14 第四節 蔗糖合成酶基因調控 14 4.1 蔗糖合成酶基因之表現具組織特異性 14 4.2 逆境對蔗糖合成酶基因表現的影響 15 4.3 糖調控蔗糖合成酶基因表現 15 第五節 糖基轉移酶家族與其結構 16 5.1 醣類代謝酵素整合型資料庫 - CAZy 16 5.2 糖基轉移酶家族與分類 16 5.3 糖基轉移酶的結構類型 18 5.4 蔗糖合成酶在糖基轉移酶家族中的分類 19 第六節 論文緣起與研究目的 19 6.1 研究緣起 19 6.2 本論文之研究目的 21 第二章 材料與方法 22 第一節 實驗材料與藥品 22 1.1 菌種 22 1.2 質體 22 1.3 實驗藥品 23 第二節 實驗儀器 23 2.1 核酸電泳設備 23 2.2 蛋白質電泳與轉印設備 23 2.3 離心機 23 2.4 恆溫水浴槽 23 2.5 分光光度計 24 2.6 其它 24 第三節 實驗方法 24 3.1 水稻蔗糖合成酶 RSuS 功能性區塊分析與結構預測 24 3.1.1 功能區塊分析 24 3.1.2 三級結構同源性分析 24 3.1.3 三級結構模擬 25 3.1.4 基質嵌合模擬 25 3.2 蔗糖合成酶活性測定法 26 3.2.1 蔗糖分解方向活性測定 26 3.2.1.1 UDPG 去氫酶酵素耦合法 26 3.2.1.2 還原糖定量法 26 3.2.2 蔗糖合成方向活性測定法: Anthrone 定量法 27 3.3 蛋白質定量與分析 28 3.3.1 蛋白質定量 28 3.2.2 蛋白質電泳 28 3.3.3 蛋白質轉印 29 3.3.4免疫染色法 29 3.4 重組蛋白質之生產與純化 30 3.4.1 重組 RSuS1 蛋白質之生產與蛋白質粗萃取 30 3.4.1.1 大量培養 30 3.4.1.2 粗抽及硫酸銨分劃 30 3.4.2 重組 RSuS3蛋白質之生產與蛋白質粗萃取 31 3.4.2.1 誘導酵母菌 Pichia pastoris 中 RSuS3 重組蛋白質的生產 31 3.4.2.2 粗抽與硫酸銨分劃 31 3.4.3 離子交換管柱層析 32 3.4.4 膠體過濾管柱層析 32 3.5 RSus3 突變株之建構與重組蛋白質生產 32 3.5.1 以定位點突變法建構 RSus3 突變株之表現質體 32 3.5.1.1 質體 DNA 小量製備 32 3.5.1.2 以聚合聚合酶連鎖反應進行定位點突變 33 3.5.1.3 PCR產物DNA以限制酶 DpnI 作用 34 3.5.1.4 質體DNA之轉形 34 (1) Competent cells之製備 34 (2) 質體之轉形 34 3.5.1.5 轉形株之檢定 35 3.5.2 酵母菌之轉形 35 3.5.2.1 直線型質體 DNA 的製備 35 3.5.2.2 酵母菌 competent cells之製備 36 3.5.2.3 電穿孔轉形 36 3.5.3 酵母菌轉形株的篩選與鑑定 36 3.5.3.1 鑑定轉形株性狀 37 3.5.3.2 目標基因插入染色體之 PCR 檢定 37 (1) 酵母菌染色體DNA的分離 37 (2) PCR檢定 38 3.5.4 酵母菌轉形株的小量表現與最佳誘導時間探討 38 3.6 水稻蔗糖合成酶之基質結合分析 39 3.6.1 平衡透析法 39 3.6.2 膠體過濾管柱層析 39 第三章 結果 41 第一節 水稻蔗糖合成酶基質結合區與活性中心之探討 41 1.1 水稻蔗糖合成酶與糖基轉移酶之多重序列比對 41 1.2 水稻蔗糖合成酶功能區塊的分析 41 1.3 水稻蔗糖合成酶蛋白質結構同源性分析 41 1.4 水稻蔗糖合成酶蛋白質結構模擬 42 1.5 水稻蔗糖合成酶蛋白質基質嵌合模擬 (substrate docking) 43 第二節 水稻蔗糖合成酶重組蛋白質之純化及活性分析 44 2.1 重組蛋白質 RSuS1 與 RSuS3 之純化 44 2.2 RSus3 突變株之建構 44 2.2.1 RSus3 突變株表現質體之建構 45 2.2.2 突變株小量表現 45 2.3 RSuS1 與 RSuS3 突變重組蛋白質之活性分析 46 第三節 水稻蔗糖合成酶與基質結合之分析 46 3.1平衡透析 47 3.2 Sephadex G25 膠體過濾管柱層析 48 3.2.1 RSuS1 與 RSuS3： 48 3.2.2 RSuS1 與 RSuS3 突變株： 48 3.2.3 添加不同基質組合： 49 第四節 水稻蔗糖合成酶 RSuS3 酵素動力學分析 49 第五節 水稻蔗糖合成酶 RSuS3 結構解析 50 5.1 蛋白質結晶與 X-ray 繞射分析 50 5.2 小角度雷射光散射分析 (Small angle X-ray scattering) 50 第四章 討論 51 第一節 水稻蔗糖合成酶具有 GT-B 摺疊結構 51 第二節 DTGGQV 及 E-X7-E motif 中之胺基酸影響水稻蔗糖合成酶之酵素活性 52 第三節 水稻蔗糖合成酶酵素催化反應機制推測為 sequential-order mechanism 52 第四節 DTGGQV 及 E-X7-E motif 中之胺基酸影響水稻蔗糖合成酶與基質的結合能力 54 第五節 Phe680 及 Glu686 影響水稻蔗糖合成酶對基質的親和力及催化反應速率 56 第五章 結論與未來展望 59 參考文獻 62 圖與表 72 附錄 104 | |
| dc.language.iso | zh-TW | |
| dc.title | 水稻蔗糖合成酶催化反應機制與蛋白質結構之探討 | zh_TW |
| dc.title | Studies on the catalytic mechanism and the structure of rice sucrose synthase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 宋賢一(Hsien-Yi Sung),楊健志(Chien-Chih Yang),楊啟伸(Chii-Shen Yang),陳佩燁,張世宗(Shih-Chung Chang) | |
| dc.subject.keyword | 蔗糖合成?,反應機制,蛋白質結構,定位點突變,同源模擬, | zh_TW |
| dc.subject.keyword | sucrose synthase,catalytic mechanism,protein structure,site-directed mutagenesis,homology modeling, | en |
| dc.relation.page | 115 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-02-13 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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