博碩士論文 107821020 詳細資訊




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姓名 傅薏華(FU, YI-HUA)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 脂肪酸特異互養棲熱菌酮醇酸還原異構酶之晶體結構及活性分析
(Thermo-active and NADH-utilizing ketol-acid reductoisomerase from Syntrophothermus Lipocalidus)
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摘要(中) 支鏈型胺基酸 (BCAA) 分別為纈胺酸 (Valine)、異白胺酸 (Isoleucine) 和白胺酸 (Leucine) ,是人體必須通過攝取食物獲得的必需胺基酸,其中催化BCAA生合成的第二步的酮醇酸還原異構酶 (Ketol-Acid Reductoisomerase, KARI) 是雙功能酶,可以進行異構化與還原兩步驟反應。過去文獻中發現大多數 KARI 對NADPH輔酶有強烈的偏好,然而使用便宜又穩定的NADH為輔酶的KARI才符合工業大規模發酵的需求。目前經由酵素活性實驗鑑定能以NADH作為輔酶的KARI 有12種,但其中僅有三種KARI與NADH結合的複合物結構被發表,且不同物種KARI與NADH作用的方式都不相同。本研究選擇研究耐熱厭氧的脂肪酸特異互養棲熱菌 (Syntrophothermus Lipocalidus)中的KARI ,因其 Sl-KARI 可能具有耐熱特性,且有機會使用較高經濟價值的NADH當輔酶。膠體過濾層析結果顯示,Sl-KARI以十二聚體的形式存在於溶液中(〜440 kDa)。從 50℃ 酵素活性實驗的結果可知,Sl-KARI可以分別使用兩種輔酶進行催化反應, 對NADH的偏好是NADPH 的1.33倍。CD光譜變溫實驗證明酵素二級結構具有熱穩定性。通過 X 光晶體繞射技術分別在 2.12Å 及 2.27 Å 的解析度下,得到兩種複合物Sl-KARI-NADH與Sl-KARI-NADPH的晶體結構。晶體結構分析顯示,Sl-KARI具有與class I KARI相同的三維摺疊,分析Sl-KARI與NADH的結合模式, 蛋白質Arg48支鏈 與腺嘌呤環形成典型的陽離子-π相互作用,而Glu58與五碳醣的2號和3號氫氧基形成氫鍵;而跟 NADPH 結合時,因 NADPH 的五碳醣上帶負電的 2´-phosphate 會和一樣是帶負電Glu58的羧基產生斥力,而拉遠了兩者的距離,因此 Sl-KARI 是藉由 Glu58 胺基酸構型改變來調控酵素使用相同活化位容納兩種不同輔酶的機制。Sl-KARI在50℃的高溫下,可以使用兩種輔酶進行催化反應,尤其是使用NADH有更好的活性,這些特性使其在厭氧或苛刻條件下,在代謝工程或工業應用上具有良好的應用潛力。
摘要(英) The branched-chain amino acid (BACC) synthetic pathway is responsible for producing of three essential amino acids, valine, isoleucine and leucine, which cannot be produced by the human body and must be obtained by ingesting food. Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme that catalyzes the second step in the biosynthesis of BCAA. Most KARI enzymes characterized in the literature show a strong preference for NADPH. However, KARIs with NADH preference are desirable in the industrial production of amino acids and biofuels. Twelve kinds of KARI have been identified as NADH-utilizing enzymes but only three structures of KARI-NADH complexes have been reported. In this study, we characterize a thermoactive Sl-KARI from thermophilic and anaerobic bacterial Syntrophothermus Lipocalidus and present its crystal structures of Sl-KARI-NADH and Sl-KARI-NADPH complexes at a 2.12Å and 2.27Å resolution. The gel-filtration result shows that Sl-KARI exists as a dodecamer in solution (~440 kDa). Enzyme activity assays reveal that Sl-KARI is a bispecific enzyme towards NAD(P)H, accepting both NADH and NADPH at a ratio of ∼1.33 at 50 ºC. The structural analysis displays that Sl-KARI possesses the same overall 3D fold as other class I KARIs. However, the conformation of a specific loop differs significantly from any other crystallized KARIs. In the NADH binding mode, the Arg48 forms a typical cation–π interaction with the adenine ring. Glu58 forms hydrogen bonds with the 2′ and 3′-OH groups of the adenosine ribose, as shown in Ua-KARI, an NADH-preferring enzyme. In the NADPH binding mode, the side chain of Arg48 undergoes the typical packing interaction against the adenine ring and forms a salt bridge with the phosphate of the cofactor. In contrast, the negatively charged side chain of Glu58 serves to repel the 2′-phosphate of NADPH. Structural alignments of NADH and NADPH complexes reveal that the coenzyme-binding loop can undergo conformational changes of Glu58 residue resulting from coenzyme replacement. Bi-cofactor utilization and the thermoactivity of Sl-KARI make it a potential candidate for metabolic engineering or industrial applications under anaerobic or harsh conditions.
關鍵字(中) ★ 酮醇酸還原異構酶
★ 脂肪酸特異互養棲熱菌
★ X 光晶體繞射
關鍵字(英) ★ KARI
★ Syntrophothermus Lipocalidus
★ NADPH
★ NADH
論文目次 中文摘要........Ⅰ
英文摘要... ....Ⅱ
誌謝............Ⅲ
目錄............Ⅳ
圖目錄..........VII
表目錄..........IX

第一章 緒論..................................................................1
1-1菌種.......................................................................1
1-1-1細菌域................................................................1
1-1-2脂肪酸特異互養棲熱菌 (Syntrophothermus lipocalidus) ..................2
1-2酮醇酸還原異構酶 (Ketol-Acid Reductoisomerase, KARI).......................3
1-2-1支鏈胺基酸 (Branched-chain amino acids, BCAA) 的生物合成途徑............3
1-2-2 KARI 的催化反應......................................................4
1-2-3 KARI 的分類及結構....................................................5
1-2-4 KARI的應用性........................................................11
1-3研究動機..................................................................13
第二章 實驗方法..............................................................15
2-1實驗架構..................................................................15
2-2基因建構..................................................................15
2-3目標蛋白質表現............................................................17
2-3-1熱休克法轉殖質體.....................................................17
2-3-2使用 E. coli BL21(DE3) 表達 Sl-KARI..................................17
2-3-3十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)........................................................................18
2-4目標蛋白質純化............................................................20
2-4-1超音波破菌...........................................................20
2-4-2離心去除變性蛋白細胞破片.............................................20
2-4-3快速蛋白質液相層析儀 (Fast Protein Liquid Chromatography) 純化.......20
2-4-3-1親和性純化法 (Immobilized Metal Affinity Chromatography) .......21
2-4-3-2 蛋白質濃縮.....................................................21
2-4-3-3膠體過濾法 (Size-exclusion Chromatography, Gel filtration) .....22
2-4-4目標蛋白濃度定量.....................................................23
2-5蛋白質長晶................................................................23
2-5-1預長晶實驗 (Pre-crystallization Test) ...............................23
2-5-2晶體條件篩選與晶體形成...............................................25
2-6 X光繞射與結構解析........................................................26
2-6-1繞射數據收集.........................................................26
2-6-2數據處理.............................................................26
2-7目標蛋白耐熱試驗..........................................................26
2-7-1圓二色光譜...........................................................26
2-8 目標蛋白質活性實驗.......................................................27
2-8-1 製備酵素的受質及其定量..............................................27
2-8-2 配製輔酶溶液........................................................27
2-8-3 酵素活性測試........................................................28
第三章 實驗結果與討論.......................................................29
3-1蛋白質純化................................................................29
3-2酵素活性實驗..............................................................32
3-2-1在50 ℃, Sl-KARI使用兩種不同輔酶,對受質2-AL的活性..................32
3-2-2 Sl-KARI對鎂離子的專一性.............................................33
3-3 Sl-KARI的熱穩定性........................................................34
3-4蛋白質長晶................................................................36
3-4-1 預長晶實驗..........................................................36
3-4-2 大量篩選及手動優化長晶條件..........................................36
3-4-2-1 Sl-KARI:NADH複合體..............................................36
3-4-2-2 Sl-KARI:NADPH複合體.............................................37
3-5 蛋白質結構分析...........................................................41
3-5-1 Sl-KARI是12聚體的Class I KARI........................................41
3-5-2 Sl-KARI 和兩種輔酶複合物的晶體結構.....................................43
3-5-3 Sl-KARI偏好NADH為輔酶並具有熱穩定性....................................46
3-5-4可使用NADH當輔酶的KARI和其輔酶複合體的結構比較..........................47
第四章 結論.................................................................49
參考文獻.....................................................................50
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指導教授 陳青諭 粘仲毅(Chin-Yu Chen Chung-Yi Nien) 審核日期 2021-1-21
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