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研究生:陳瑞娟
研究生(外文):Jui-Chuan Chen
論文名稱:以化學修飾法與定點突變法研究無乳鏈球菌之唾液酸合成酶內必需的半胱胺酸與精胺酸
論文名稱(外文):Chemical Modification and Site-directed Mutagenesis of Cys and Arg of the Sialic Acid Synthase from Streptococcus agalactiae
指導教授:林俊宏林俊宏引用關係
指導教授(外文):Chun-Hung Lin
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:64
中文關鍵詞:唾液酸合成酶
外文關鍵詞:sialic acid synthase
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B型無乳鏈球菌(Group B Streptococcus agalactiae)含唾液酸的莢膜,能避免補體C3b的沉澱或巨噬細胞的吞噬,使菌體可經由垂直感染造成新生兒敗血症或腦膜炎。莢膜多醣的產生及其結構的完整性,已被報導為該菌的致病毒性因子。而唾液酸位於莢膜上寡醣單元(NeuAc-��2,3-Gal-��1,4-GlcNAc-��1,3-Gal-��1,4-Glcl)的末端,主要由唾液酸合成酶(E.C.4.1.3.19) (簡稱為SaNeuB)負責生成,是該菌體能夠造成致病的重要因素之一,顯見此酵素的重要性。
SaNeuB含341個胺基酸,分子量約為39 kDa。當二價金屬離子,如鎂、錳、鈷或鎳離子存在時,可進行催化反應,結合受質phospho(enol)pyruvate (PEP)及N-acetylmannoseamine (ManNAc) 進行不可逆的縮合反應,產生唾液酸並釋放一個無機磷酸根。了解SaNeuB活化中心內參與催化,或與受質結合有關的胺基酸,將有助於此酵素抑制物的設計,進而達到抑菌效果。故本研究利用專一性化學修飾法及定點突變法,針對活性中心內可能參與金屬螯合的半胱胺酸(Cys)及可能與PEP受質結合的精胺酸(Arg)進行研究。
以IAA(Cys的修飾試劑)及PGO(Arg的修飾試劑)分別作用於SaNeuB,發現其活性隨試劑作用時間及濃度增加而降低,最後完全喪失活性;若先以PEP/Mn2+進行受質保護,則酵素被IAA及PGO修飾時,活性分別可維持對照組 (不加修飾試劑) 的60%及40%,進一步證實Cys及Arg的確位於活性區中。另以DTNB測定Cys之sulfhydryl group,未測到雙硫鍵的存在,也證明酵素失活並非由雙硫鍵的破壞所致。
為了更清楚了解SaNeuB中那一個Cys及Arg參與在活性區內,以定點突變法將酵素內的3個Cys及9個Arg突變為Ala,分別測定酵素比活性,其中突變株C10A,C169A及R83A活性剩下約20%左右,而R301A及R277A則完全失去活性。以圓二色偏光光譜儀測定這些突變株之二級結構,結果與原型SaNeuB並沒有很大的差別,顯示酵素失活與結構改變無關。綜合化學修飾及定點突變的結果,與受質相似酵素KDO8P合成酶及DAH7P合成酶在胺基酸區塊序列的比對,我們推測:Cys10,Cys169可能與金屬的螯合有關;而Arg277,Arg301則可能與PEP phosphate group結合有關。日後希望配合蛋白質晶體結構的解析,以確定這些胺基酸確切的功能與角色。
Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. The production of a type-specific capsular polysaccharide (CPS) is essential to the virulence of this pathogen and enables the bacteria to evade host immue defenses. As a key component of the capsular polysaccharide, sialic acid (or N-acetylneuraminic acid) is ��2,3-linked to galactose to form a disaccharide that is pending from the repeating trisaccharyl backbone. Sialic acid synthase (E.C. 4.1.3.19, NeuB) is the important enzyme which is responsible for sialic acid biosynthesis.
The sialic acid synthase from Streptococcus agalactiae (abbreviated as SaNeuB) consists of 341 amino acids corresponding to the molecular mass of 39 kDa. The enzyme catalyzes the irreversible aldol condensation of PEP and ManNAc to form sialic acid and is active only in the presence of a divalent metal ion, such as Mg2+, Mn2+, Co2+ or Ni2+. Understanding the residues involved in catalysis or substrate binding is a prerequisite to design enzyme inhibitors and develop anti-bacterial reagents. This work presents the modification of Cys and Arg residues by using specific chemical modifiers and site-directed mutagenesis as the two residues probably participate in the metal coordination and the binding of PEP, respectively.
IAA (a cysteine modifier) and PGO (an arginine modifier) were utilized to inactivate SaNeuB in a time- and dose-dependent manner, revealing that Cys and Arg are important to enzyme activity. The substrate protection with PEP and Mn2+, remained around 60% (for IAA inactivation) and 40% (for PGO inactivation) of the control, respectively, supporting that Cys and Arg are in or near the active site. DTNB was used to carry out the titration of sulfhydryl group. No disulfide bond was found in SaNeuB, based on the DTNB titration study of the sulfhydryl group. As a consequence, the activity loss due to the cysteine modifier is not associated with the disruption of disalfide bond.
Furthermore, three Cys�_Ala and nine Arg �_ Ala mutants were generated by site-directed mutagenesis. The mutants C10A, C169A and R83A retain 20% activity, whereas R277A and R301A mutants were virtually inactive. A comparison of the circular dichroism spectra of the wild-type and all the mutants indicated that there was no significant difference. Although the kinetics studies showed no obvious changes in the Km of PEP and ManNAc, the results obtained from the chemical modification and site-directed mutagenesis studies implied that some of the Cys and Arg residues are essential to enzyme catalysis. According to the comparison of the conserved sequence with KDO8P synthase and DAH7P synthase, we suggest that Cys10 and C169 may play an important role in metal coordination, Arg277 and Arg301 may involve in the binding of phosphate group of PEP.
目 錄

謝誌 I
目錄 II
圖表附錄 VI
縮寫表 IX
中文摘要 X
英文摘要 XI

壹. 緒論 1
一. 唾液酸 (sialic acid) 及Streptococcus agalactiae莢膜多醣 1
1. 唾液酸的結構與唾液酸醣鏈分子 1
2. 唾液酸的功能 2
3. Neu5Ac的合成與代謝 2
4. Streptococcus agalactiae 含唾液酸之莢膜多醣 3
二. 唾液酸合成酶的研究概況 5
1. E.coli K1唾液酸合成酶 5
2. N. mengitidis唾液酸合成酶 6
3. Campylobacter jejuni唾液酸合成酶 6
4. Human唾液酸合成酶 7
5. Rat唾液酸合成酶 7
6. Drosophila malanogaster唾液酸合成酶 8
7. Streptococcus agalactiae 唾液酸合成酶 8
8. 唾液酸合成酶一般特性及動力學分析比較 9

三. 唾液酸合成酶及其相關酵素對活性區 (active site) 胺基酸的研究 12
1. KDO8P synthase 12
2. DAH7P synthase 13
3. DAH7P synthase中對Cys residues的研究 14
4. KDO8PS中對Cys residues的研究 15
5. 老鼠肝臟的唾液酸合成酶中對Cys residues的研究 16
6. Streptococcus agalactiae的唾液酸合成酶中對Arg residues的研究 16
7. 以定點突變法對大腸桿菌K1的唾液酸合成酶的活性區的研究 16
四. 實驗動機 18
貳. 材料與方法 19
一. 實驗材料 19
1. 藥品 19
1.1. 一般化學藥品 19
1.2. 限制酶與實驗相關酵素 19
2. 菌株及載體 19
二. 實驗之儀器設備 19
三. 實驗方法 20
1. 基因選殖 (Gene cloning) 20
1.1. 小量質體DNA的抽取 (Isolation the plasmid DNA) 20
1.2. DNA瓊脂明膠電泳 (DNA agarose gel electrophoresis) 21
1.3. 膠體中DNA之回收與純化 (DNA recovery and purify) 21
1.4. 兩步驟聚合酶連鎖反應 (Two step PCR) 21
1.5. 限制酶切割 (Restriction enzyme digestion) 22
1.6. 載體與基因之結合 (DNA ligation) 23
1.7. 以氯化鈣法勝任細胞的製備(competent cell preparation) 23
1.8. DNA轉型作用 (DNA transformation) 23
1.9. 選殖基因之確認 24
2. 蛋白質純化與鑑定 (Protein purify and identification) 24
2.1. 蛋白質之誘導與表現 (Protein induction and expression) 24
2.2. 蛋白質粗抽取液之取得 (Protein clued extract gathering) 24
2.3. 蛋白質之純化 25
2.3.1. 硫酸鎳金屬親和性管柱分析 25
2.3.2. 離子交換管柱分析 25
2.3.3. 蛋白質濃縮及去鹽去金屬 26
2.3.4. 蛋白質之保存 26
2.4. 蛋白質之定量 26
2.4.1. Bradford定量 26
2.4.2. 吸光法定量蛋白質 26
2.5. 蛋白質聚丙醯胺膠體電泳 (SDS-PAGE) 之分析 27
2.6. 蛋白質分子量以質譜儀 (MASS) 確認 28
2.7. 蛋白質N端(N-terminal)序列分析 28
3. 酵素活性的測定 29
3.1. 酵素反應液配製 29
3.2. 酵素活性測定方法-TBA (Thiobarbituric acid) assay 29
3.3. 唾液酸標準曲線 30
3.4. 酵素比活性測定 31
3.5. 酵素動力學測定 31
4. 圓二色偏光光譜(Circular dichroism,CD)測定 31
5. 化學修飾試劑針對酵素內半胱胺酸之抑制實驗 32
5.1. 專一性化學修飾試劑之抑制實驗 32
5.2. 不同濃度之IAA於不同時間下作用於SaNeuB之實驗 32
5.3. 受質保護實驗 (Substrate protection test) 32
5.4. DTNB titration 33
6. 化學修飾試劑針對酵素內精胺酸之抑制實驗 33
6.1. 專一性化學修飾試劑之抑制實驗 33
6.2. 不同濃度之PGO於不同時間下作用於SaNeuB之實驗 33
6.3. 受質保護實驗 (Substrate protection test) 34
參. 實驗結果 35
一. 質體建構之結果 35
二. 蛋白質表現與純化之結果 35
三. 蛋白質N端定序之結果 36
四. 唾液酸合成酶等電點梯度電泳圖 36
五. 半胱胺酸單點突變之建構 36
六. 精胺酸單點突變之建構 37
七. 突變之唾液酸合成酶活性及動力學分析 37
1. 半胱胺酸突變之唾液酸合成酶比活性及動力學分析 37
2. 精胺酸突變之唾液酸合成酶比活性及動力學分析 38
八. 圓二色偏光光譜分析-野生型及突變型之唾液酸合成酶 39
九. 野生型及突變型唾液酸合成酶分子量測定之結果 39
十. 半胱胺酸化學修飾試劑作用於唾液酸合成酶之結果 39
十一.精胺酸化學修飾試劑作用於唾液酸合成酶之結果 40
十二.受質保護實驗之結果 40
十三.DTNB titration之結果 40
肆. 結果與討論 42
一. Streptococcus agalactiae唾液酸合成酶之一般特性 42
1. 溫度的影響 42
2. 酸鹼度的影響 42
3. 二價金屬離子的影響 42
4. 分子量及等電點 43
5. N-端序列分析 43
二. 半胱胺酸在SaNeuB中角色之探討 44
1. 化學修飾試劑作用後影響酵素活性之探討 44
2. 胺基酸定點突變後影響酵素活性之探討 46
3. 半胱胺酸在唾液酸合成酶中角色之臆測 47
4. 以電子噴灑式質譜技術鑑定出與化學修飾試劑作用的半胱胺酸 47
三. 精胺酸在SaNeuB中角色之探討 49
 1. 化學修飾試劑作用後影響酵素活性之探討 49
2. 胺基酸定點突變後影響酵素活性之探討 50
 3. 精胺酸在唾液酸合成酶中角色之臆測 50
伍. 未來展望 52
陸. 參考文獻 53
表次
圖次
附錄目次

表次
表一、唾液酸的醣鏈分子及其分佈於細胞的位置 1
表二、不同物種中唾液酸合成酶之特性比較 10 
表三、不同物種中唾液酸合成酶之比活性及酵素動力學比較 11
表四、菌株及質體命名對照表 58
表五、引子序列表 59
表六、野生型及突變型唾液酸合成酶之活性表 60
表七、野生型及突變型唾液酸合成酶之酵素動力學分析表 61
表八、唾液酸合成酶相似酵素-活性區相關胺基酸分析表 62
表九、唾液酸合成酶N端序列析表 62
表十、唾液酸合成酶及其突變酵素之二級結構比例分析 63
表十一、DTNB titration測定唾液酸合成酶及突變酵素之半胱胺酸個數 64
表十二、以高或低濃度之PEP與其相似物BrPy作用於酵素後對酵素活性及半胱胺酸標計的個數表 64

圖次
圖一、唾液酸的生合成、代謝及修飾 3
圖二、B型無乳鏈球菌血清型Ia及III之寡醣單元 4
圖三、建構選殖之質體 65
圖四、建構定點突變基因 66
圖五、Streptococcus agalactiae唾液酸合成酶基因與胺基酸序列 67
圖六、DNA瓊脂明膠電泳圖 68
圖七、原核及真核生物唾液酸合成酶序列比對 69
圖八、圓二色偏光光譜分析唾液酸合成酶及其突變酵素 70
圖九、親和性層析法(a)及離子交換層析法(b)純化唾液酸合成酶 71
圖十、蛋白質電泳圖-Streptococcus agalactiae NeuB 之純化 72
圖十一、不同濃度之IAA在不同時間下作用於唾液酸合成酶之結果 73
圖十二、不同濃度之PGO在不同時間下作用於唾液酸合成酶之結果 74
圖十三、受質保護實驗之結果 75
圖十四、DTNB titration之結果 76
圖十五、唾液酸合成酶等電點梯度電泳圖 77
圖十六、唾液酸合酶N端序列分析圖 78


附錄目次

附錄一、培養基、抗生素、緩衝液配方 81
附錄二、SaNeuB與相似酵素KDO8P synthase及DAH7P synthase之反應式 82
附錄三、TBA (Thiobarbituric Acid)反應機構 83
附錄四、化學修飾試劑與半胱胺酸殘基之作用機制 84
附錄五、化學修飾試劑與精胺酸殘基之作用機制 85
附錄六、KDO8P合成酶活性區之結構 86
附錄七、DAH7P合成酶活性區之結構 87
附錄八、定點突變之基因序列 88
附錄九、突變型唾液酸合成酶之質譜分析 91
附錄十、以電子噴灑式質譜技術鑑定出與BrPy作用的半胱胺酸之質譜圖 105
陸、參考文獻
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4.Ferrero, M. A., Reglero, A., Fernandez-Lopez, M., Ordas, R., and Rodriguez-Aparicio, L.B. (1995) N-Acetyl-D-neuraminic acid lyase generates the sialic acid for colominic acid biosynthesis in Escherichia coli K1. Biochem. J. 317, 157-165.

5.Rodriguez-Aparicio, L.B., Ferrero, M.A., and Reglero, A. (1995) N-Acetyl-D-neuraminic acid synthesis in Escherichia coli K1 occurs through condensation of N-acetyl-D-mannosamine and pyruvate. Biochem.J. 308, 501-505.

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