硫化葉菌屬中耐熱酮醇酸還原異構酶之結構性及功能性分析

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周志懋(Chih-Mao Chou) 查詢紙本館藏

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生命科學系

論文名稱

硫化葉菌屬中耐熱酮醇酸還原異構酶之結構性及功能性分析
(Structural and Functional Analysis of the Thermotolerant Ketol-Acid Reductoisomerase (KARI) from Sulfolobus species)

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摘要(中)

酮醇酸還原異構酶是參與支鏈型胺基酸生合成的重要酵素，這個酵素只會出現在細菌、古生菌、真菌和植物中，動物則沒有。此酵素催化兩步驟反應，第一步為鎂離子專一性之烷基轉移，在緊接著進行具有 NAD(P)H 和二價金屬 (Mg2+、Mn2+ 及 Co2+) 依賴性之還原反應。大多數被發現的酮醇酸還原異構酶偏好 NADPH，這是因為一般生物是在有氧的環境下生存。在許多文獻中，為了要讓細菌能夠發酵生成支鏈型胺基酸或是將葡萄糖轉化為醇類，利用基因工程的方式將酮醇酸還原異構酶之輔因子專一性改成 NADH。在我們的研究中，從生存在高溫且強酸性之極端環境中的硫化葉屬古生菌中，取得該酵素。在嗜酸熱硫化葉菌中取得 Sac-ilvC；在硫磺礦硫化葉菌中取得 Sso-ilvC-1、Sso-ilvC-2 和 Sso-ilvC-3。在利用 X-ray 蛋白質晶體學以及低溫電子顯微鏡這兩個技術，得到 Sac-ilvC 和 Sso-ilvC-2 之原子級別結構與 Sso-ilvC-3 之奈米級別結構。再利用光譜學技術對其生化特性進行分析。我們發現 Sac-ilvC 與 Sso-ilvC-1 在序列及特性上都較為接近並且對輔因子具有雙專一性；而 Sso-ilvC-2 與 Sso-ilvC-3 兩者較為相似，會形成十二聚體並且具有 NADH 專一性。酮醇酸異構還原酶聚合化會影響其結構穩定性並提高耐熱能力，越是容易聚合之酵素其耐熱能力就越好。至於，硫磺礦硫化葉菌會擁有三個編碼基因的現象，目前推測為該菌若遭遇到更嚴厲之環境，更高溫且含氧量更低，則需要會使用 NADH 且更耐熱之蛋白質。

摘要(英)

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of branched-chain amino acids, which is present in plants, fungi, archaea and bacteria, but not in animals. This enzymatic reaction involves an Mg2+-dependent alkyl migration followed by a NADPH- and divalent metal ions-dependent reduction of the 2-keto group. The most of the KARIs characterized to date have been shown to prefer the NADPH cofactor to NADH since these nearly were obtained from the organisms living in aerobic environment. In many case, KARIs switching the cofactor preference to NADH are desirable for industrial applications by genetic engineering, including anaerobic fermentation to produce branched-chain amino acids and some kinds of alcohol. In our study, we obtained four different KARI from Sulfolobus species which are extremophiles living in harsh environments with high temperature and low pH value. Sac-ilvC was gained from Sulfolobus acidocaldarius and Sso-ilvC-1, Sso-ilvC-2 and Sso-ilvC-3 were gained from Sulfolobus solfataricus. Here, we determined the structure of Sso-ilvC-2 by X-ray crystallography (2.5 Å) and cryo-EM (6 Å). The Sso-ilvC-2 forms dodecamer by assembling six dimers as tetrahedral shape. Sso-ilvC-2 and Sso-ilvC-3 are putative NADH-dependent KARI. We supposed S. solfataricus has three KARI encoding genes, which induced the thermoresistan protein that consume NADH when it encountered the much more extreme environment, higher temperature and lower level of oxygen content.

關鍵字(中)

★ 酮醇酸還原異構酶

關鍵字(英)

★ Ketol-Acid Reductoisomerase

論文目次

中文摘要 i
英文摘要 ii
致謝 iii
目錄 iv
表目錄 vii
圖目錄 viii
第一章、緒論 1
1-1 硫化葉菌屬 1
1-1-1 古生菌 (Archaea) 1
1-1-2 嗜酸熱硫化葉菌 (Sulfolobus acidocaldarius) 6
1-1-3 硫磺礦硫化葉菌 (Sulfolobus solfataricus) 7
1-2 酮醇酸還原異構酶 (Ketol-Acid Reductoisomerase, KARI) 8
1-2-1 介紹 8
1-2-2 在細胞內位置 9
1-2-3 催化特性 10
1-2-4 抑制機制 10
1-2-5 結構及其分類 11
1-2-6 應用性 14
1-3 研究動機 16
第二章、材料與儀器 19
2-1 材料 19
2-2 儀器 22
第三章、實驗方法與流程 23
3-1 建構目標蛋白質基因及其載體 23
3-1-1 核酸引子設計 23
3-1-2 準備嗜熱古生菌硫化葉菌株之全基因體萃取 25
3-1-3 核酸聚合酶鏈鎖反應擴增目標基因 25
3-1-4 利用膠體電泳確認目標基因片段 27
3-1-5 核酸純化 27
3-1-6 限制酶切割反應 (Digestion) 28
3-1-7 連接反應 (Ligation) 28
3-1-8 勝任細胞 (Competent Cell) 製備 29
3-1-9 熱休克法 30
3-1-10 菌落核酸聚合酶鏈鎖反應 (Colony PCR) 30
3-1-11 核酸定序 31
3-2 蛋白質表現 33
3-2-1 轉型至表現菌株 33
3-2-2 表現時程測試 (Time Course) 33
3-2-3 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 34
3-2-4 大量表現蛋白質 35
3-3 目標蛋白質純化 37
3-3-1 破壞細菌細胞 37
3-3-2 離心去除變性蛋白及細胞破片 38
3-3-3 加熱處理 (Heat treatment) 39
3-3-4 固定化金屬親和性層析 (Immobilized Metal Affinity Chromatography) 39
3-3-5 蛋白質濃縮 41
3-3-6 分子篩層析 (Size Exclusion Chromatography) 42
3-3-7 目標蛋白質濃度定量 43
3-4 利用 X-ray 晶體繞射技術解析蛋白質結構 45
3-4-1 蛋白質結晶 45
3-4-2 預結晶試驗 46
3-4-3 蛋白質結晶條件篩選 47
3-4-4 繞射數據收集 49
3-4-5 解決相位問題/結構建立及其優化 50
3-5 利用低溫電子顯微鏡技術解析蛋白質結構 51
3-5-1 冷凍樣品製作 51
3-5-2 收集低溫電顯影像數據 52
3-5-3 單分子影像重建及其優化 54
3-6 目標蛋白質耐熱及耐酸性試驗 56
3-6-1 圓二色光譜 (CD Spectroscopy) 56
3-6-2 利用圓二色光譜進行耐熱試驗 57
3-6-3 利用圓二色光譜進行耐酸試驗 59
3-7 目標蛋白質活性測試 60
3-7-1 製備酵素受質及其定量 60
3-7-2 酵素活性測試 61
第四章、結果 62
4-1 建構攜帶目標蛋白質基因之載體 62
4-2 蛋白質表現測試 63
4-3 蛋白質純化 66
4-3-1 加熱處理之結果 66
4-3-2 蛋白質純化 68
4-4晶體繞射之蛋白質結構 73
4-4-1 結晶條件篩選結果 73
4-4-2 結晶條件之優化 74
4-4-3 蛋白質結構 79
4-5電顯技術重建之蛋白質結構 82
4-6 耐熱及耐酸性試驗之結果 87
4-6-1 圓二色光譜單位及轉換 87
4-6-2 耐熱實驗 89
4-6-3 耐酸實驗 92
4-7 活性測試之結果 94
4-7-1 受質製備 94
4-7-2 酵素活性測試結果 94
第五章、討論 98
5-1 兩種結構模型 98
5-2 輔因子專一性 101
5-3 耐熱性質 105
5-4 生理意義 106
參考文獻 107

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指導教授

陳青諭(Chin-Yu Chen)

審核日期

2016-12-23

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