嗜酸熱硫化葉菌酮醇酸還原異構酶與輔酶共晶體結構及活性分析

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研究生：	陳奕勳 Yi-Hsun Chen
論文名稱：	嗜酸熱硫化葉菌酮醇酸還原異構酶與輔酶共晶體結構及活性分析 Cofactor bi-specificity in Sulfolobus acidocaldarius Ketol-Acid Reductoisomerase as revealed by two crystal structures and enzyme activity assays
指導教授：	陳青諭 Chin-Yu Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2020
畢業學年度：	108
語文別：	中文
論文頁數：	87
中文關鍵詞：	嗜酸熱硫化葉菌、酮醇酸還原異構酶、蛋白質結晶學、輔酶雙特異性
外文關鍵詞：	Sulfolobus acidocaldarius, Ketol-acid reductoisomerase, X-ray crystallography, Cofactor bi-specificity
相關次數：	點閱：20 下載：0
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中文摘要
酮醇酸還原異構酶(KARI) 是支鏈胺基酸(BCAA) 生物合成途徑中的第二個酵素，
為雙功能酶可進行兩步驟的催化反應。第一步進行鎂離子專一性的異構反應，隨後為輔
酶NAD(P)H 與鎂離子或其他二價金屬離子(錳離子或鈷離子) 依賴性的還原反應。在
先前研究結果，已得知嗜酸熱硫化葉菌的KARI (Sac-KARI) 具有熱穩定性與輔酶雙專
一性，可以利用NADPH 與NADH 兩種輔酶進行催化反應，且相對偏好於NADPH 。
在本次研究中，我們利用X-ray 蛋白質晶體繞射，解析出Sac-KARI-NADPH 與
Sac-KARI-NADH 兩種複合物晶體結構，解析度分別為2.72 Å 與 1.68 Å 。分析
Sac-KARI 與兩種輔酶複合物結構，發現於活化位β2αB-loop 構形並無明顯改變，但在
Sac-KARI-NADH 結構中發現來自母液的磷酸根佔據了相對於Sac-KARI-NADPH 結
構中NADPH 的2 端磷酸根基團的位置。並於高溫的酵素動力學實驗，比較不同的緩
衝液(HEPES 與磷酸鹽) 對Sac-KARI 相對活性的影響。其結果表示當酵素在使用
NADH 時，在緩衝液含有磷酸根離子環境中，會有相對較高的活性。證實了磷酸根或
是硫酸根離子是可以幫助Sac-KARI 在使用NADH 時的催化活性。

Abstract
Ketol-acid reductoisomerase (KARI) is the second enzyme in branched chain amino acid
(BCAA) biosynthetic pathway. The catalytic reaction of this bi-functional enzyme is consisted
of two steps, including Mg2+-dependent alkyl migration, followed by the
NAD(P)H-dependent reduction reaction. In the previous study, we found that KARI from
Sulfolobus acidocaldarius (Sac-KARI) is a thermostable and bi-cofactor-utilizing enzyme. In
this study, we determined two crystal structures of Sac-KARI-NADPH (2.72 Å) and
Sac-KARI-NADH (1.68 Å) complexes. The crystal structural analysis shows that R49
undergoes the typical π-cation stacking interaction against the adenine ring and forms a salt
bridge with the 2´-phosphate of the NADPH. The S53 forms H bonds both with 2´-phosphate
and 3´-OH of the NADPH. The R49 and S53 make similar contacts with NADH, however, the
phosphate ions mimic the 2´-phosphate of NADPH in the cofactor binding pocket. The
enzyme activity assays further confirm that the Sac-KARI has higher activity in the phosphate
than that in the HEPES buffer at 50ºC by using NADH as a cofactor. On the other hand, the
optimum pH for Sac-KARI activity is in the pH range of 7-7.5 at 50ºC.

目錄
中文摘要 ....................................................................................................................................I
英文摘要 .................................................................................................................................. II
致謝.........................................................................................................................................III
目錄 .........................................................................................................................................IV
圖目錄 ................................................................................................................................... VII
表目錄 .....................................................................................................................................IX
第一章、緒論 ...........................................................................................................................1
1-1 嗜酸熱硫化葉菌(Sulfolobus acidocaldarius) .......................................................1
1-2 酮醇酸還原異構酶....................................................................................................2
1-2-1 支鏈胺基酸(Branched-Chain Amino Acids, BCAA) 的生物合成途徑.....2
1-2-2 酮醇酸還原異構酶的催化機制....................................................................3
1-2-3 酮醇酸還原異構酶的分類方式與結構介紹................................................3
1-2-4 相關應用........................................................................................................6
1-3 研究動機....................................................................................................................9
第二章、實驗內容與方法.....................................................................................................10
2-1 建構目標蛋白質基因(Sac-KARI) 於載體(pET-21a) ......................................10
2-1-1 從嗜酸熱硫化葉菌全基因體中萃取出目標基因......................................10
2-1-2 聚合酶連鎖反應(Polymerase Chain Reaction, PCR) 引子設計.............. 11
2-1-3 限制酶切割反應(Digestion) ....................................................................13
2-1-4 連接反應(Ligation) ..................................................................................14
2-1-5 勝任細胞(Competent cell) ........................................................................14
2-1-6 轉形作用(Transformation) .......................................................................15
2-1-7 菌落聚合酶連鎖反應(Colony PCR) .......................................................15
2-1-8 利用瓊脂糖凝膠電泳(Agarose gel electrophoresis) 確認目標基因.......17
2-1-9 核酸定序檢測(Sequencing) .....................................................................18
2-2 蛋白質表現(Protein expression) .........................................................................19
2-2-1 篩選表現條件之時程實驗(Time Course) ...............................................19
2-2-2 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE) .............................19
2-2-3 大量表現蛋白質..........................................................................................21
2-3 蛋白質純化(Protein purification) ........................................................................22
2-3-1 利用超音波細胞破碎儀(Ultrasonic Processor) 破壞細菌細胞...............22
2-3-2 加熱處理......................................................................................................22
2-3-3 離心及過濾去除沉澱蛋白質......................................................................23
2-3-4 固定化金屬親和性層析法..........................................................................23
2-3-5 濃縮蛋白質(Concentrate protein) ............................................................25
2-3-6 凝膠過濾法(Gel filtration, Size Exclusion Chromatography) .................26
2-4 利用X-ray 蛋白質晶體繞射解析結構..................................................................27
2-4-1 蛋白質結晶(Crystallization) ....................................................................27
2-4-2 預長晶測試實驗(Pre-crystallization Test, PCT) ......................................28
2-4-3 高通量篩選蛋白質長晶條件......................................................................30
2-4-4 收集晶體繞射數據(Data collection) .......................................................31
2-4-5 利用電腦軟體分析數據(Refinement) .....................................................32
2-5 蛋白質活性測試......................................................................................................34
2-5-1 輔酶 (Coenzyme) 製備...............................................................................34
2-5-2 受質(Substrate) 製備.................................................................................34
2-5-3 酵素活性實驗..............................................................................................35
第三章、實驗結果.................................................................................................................36
3-1 核酸定序檢測..........................................................................................................36
3-2 蛋白質表現..............................................................................................................37
3-3 蛋白質純化..............................................................................................................38
3-4 蛋白質晶體繞射之結構解析..................................................................................40
3-4-1 蛋白質結晶條件..........................................................................................40
3-4-2 蛋白質結構解析..........................................................................................46
3-5 蛋白質活性測試......................................................................................................51
3-5-1 Sac-KARI 對鈣離子利用之酵素活性測試.................................................51
3-5-2 Sac-KARI 在不同pH 值中之酵素活性測試............................................53
3-5-3 Sac-KARI 在不同緩衝液中之酵素活性測試.............................................56
第四章、討論.........................................................................................................................58
4-1 比較Sso-KARI 與Sac-KARI 在不同pH 值下的酵素活性變化...................58
4-2 分析不同KARI 活化位上之胺基酸側鏈使用輔酶催化之策略........................59
4-3 Sac-KARI 活化位在催化過程中的誘導契合機制................................................63
第五章、結論.........................................................................................................................67
參考文獻 .................................................................................................................................68
附錄 .........................................................................................................................................71

圖目錄
圖 1、三域演化樹。...............................................................................................................1
圖 2、支鏈胺基酸的生物合成途徑。...................................................................................2
圖 3、KARI 兩步驟的催化反應。.....................................................................................3
圖 4、Sso-KARI-NADPH-CPD 之活化位上的結構細節。..............................................5
圖 5、對KARI 的β2αB-loop 序列比對與分類歸納。....................................................6
圖 6、基因工程大腸桿菌的生物合成途徑以生產異丁醇。...............................................7
圖 7、在KARI 催化反應中的受質、中間物及其類似物。.............................................8
圖 8、載體pET-21a 圖譜之重組與表現區段。................................................................ 11
圖 9、限制酶剪切反應之序列與切口處， NdeI (左) ， XhoI (右) 。..........................13
圖 10、細菌生長曲線圖。...................................................................................................21
圖 11、Ni-NTA 與目標蛋白質上的His-tag 結合示意圖。.........................................24
圖 12、親和性層析之蛋白質純化流程示意圖。...............................................................24
圖 13、凝膠過濾法之原理示意圖。...................................................................................26
圖 14、蛋白質結晶過程之相變圖。...................................................................................28
圖 15、蒸氣擴散法(座式) 示意圖。.................................................................................28
圖 16、輕微沉澱(Light Precipitate) 與嚴重沉澱(Heavy Amorphous Precipitate)。.....29
圖 17、單孔式96 孔坐式養晶盤。...................................................................................30
圖 18、NAD(P)H 氧化還原形式之340 nm 特徵峰的變化。.......................................35
圖 19、Sac-KARI 蛋白質表現之實驗步驟圖。..............................................................37
圖 20、Sac-KARI 親和性層析法純化結果。..................................................................38
圖 21、Sac-KARI 膠體過濾法純化結果。......................................................................39
圖 22、以Coomassie blue 染色15% SDS-PAGE 來分析Sac-KARI 的純度。..........39
圖 23、Sac-KARI-NADPH 複合物及其活化位的結構。...............................................49
圖 24、Sac-KARI-NADH 複合物及其活化位的結構。.................................................50
圖 25、Sac-KARI 對5 mM 鎂離子之酵素活性測試。.................................................52
圖 26、Sac-KARI 對10 mM 鈣離子之酵素活性測試。...............................................52
圖 27、Sac-KARI 對20 mM 鈣離子之酵素活性測試。...............................................53
圖 28、Sac-KARI 對30 mM 鈣離子之酵素活性測試。...............................................53
圖 29、在25℃ ，不同pH 值的緩衝液中Sac-KARI 利用兩種輔酶的相對活性。.54
圖 30、在50℃ ，不同 pH 值的緩衝液中Sac-KARI 利用兩種輔酶的相對活性。.55
圖 31、在25℃ ，三種不同緩衝液中 Sac-KARI 利用兩種輔酶的相對活性。..........56
圖 32、在50℃ ，三種不同緩衝液中 Sac-KARI 利用兩種輔酶的相對活性。..........57
圖 33、在55℃ ，不同pH 值的緩衝液中Sso-KARI 利用兩種輔酶的相對活性。.58
圖 34、Sac-KARI-NAD(P)H 複合物之β2αB-loop 結構疊圖。 ...................................59
圖 35、Ia-KARI-NADPH 複合物之活化位結構。..........................................................61
圖 36、Ia-KARI-NADH 複合物之活化位結構。............................................................61
圖 37、Ia-KARI-NAD(P)H 複合物之β2αB-loop 結構疊圖。......................................62
圖 38、Sso-KARI-NAD(P)H-CPD 複合物之β2αB-loop 結構疊圖。 ..........................63
圖 39、Sac-KARI-NAD(P)H 對Sac-KARI 之N domain 比對。.................................65
圖 40、Sac-KARI-NAD(P)H 對Sac-KARI 之C domain 比對。.................................65

表目錄
表 1、比較Class I 與Class II KARI 的結構組成。.........................................................4
表 2、對輔酶雙特異性或對輔酶NADH 偏好的KARI 。..............................................9
表 3、Sac-KARI 聚合酶鏈鎖反應配方。........................................................................12
表 4、Sac-KARI 聚合酶鏈鎖反應步驟。........................................................................12
表 5、限制酶剪切反應配方。.............................................................................................13
表 6、接合酶黏合反應配方。.............................................................................................14
表 7、Sac-KARI 菌落聚合酶鏈鎖反應配方。................................................................16
表 8、Sac-KARI 菌落聚合酶鏈鎖反應步驟。................................................................16
表 9、50X TAE Buffer 配製方法。....................................................................................17
表 10、15% SDS-PAGE 膠片配方與不同分子量的蛋白質所適合之膠體濃度。........20
表 11、SDS-PAGE 實驗所使用之緩衝液配方。.............................................................20
表 12、PCT 試劑成分。....................................................................................................29
表 13、PCT 結果與操作建議。........................................................................................29
表 14、Sac-KARI-NADPH-CPD 長晶條件編號與照片。...............................................40
表 15、Sac-KARI-NADPH 長晶條件編號與照片 (1)。................................................41
表 16、Sac-KARI-NADPH 長晶條件編號與照片 (2)。................................................42
表 17、Sac-KARI-NADH 長晶條件編號與照片(1)。...................................................43
表 18、Sac-KARI-NADH 篩選結晶之蛋白質樣品條件與結果(2) ~ (8)。..................44
表 19、Sac-KARI-NADH 長晶條件編號與照片(6)。...................................................44
表 20、手動養晶微調[10.2]-96 結晶條件。....................................................................45
表 21、Sac-KARI-NADH 長晶條件編號與照片(8)。...................................................45
表 22、X-ray 繞射數據收集與處理之參數以及上機晶體的照片。..............................47
表 23、在25℃ ，不同pH 值的緩衝液中Sac-KARI 利用兩種輔酶的酵素專一活性。
.................................................................................................................................................54
表 24、在50℃ ，不同pH 值的緩衝液中Sac-KARI 利用兩種輔酶的酵素專一活性。
.................................................................................................................................................55
表 25、在25℃ ，三種不同緩衝液中 Sac-KARI 利用兩種輔酶的酵素專一活性。..56
表 26、在50℃ ，三種不同緩衝液中 Sac-KARI 利用兩種輔酶的酵素專一活性。..57
表 27、比較不同物種KARI 與配體結合前後的構形變化。.........................................66
                                

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