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研究生:林政鋒
研究生(外文):Lin,Cheng-Feng
論文名稱:人類重組四羥基苯基丙酮酸雙氧合酶之羧酸端催化角色之研究
論文名稱(外文):The catalytic role of C-terminus in human 4-hydroxyphenylpyruvate dioxygenase
指導教授:李惠珍李惠珍引用關係
指導教授(外文):Hwei-Jen Lee
口試委員:周慰遠洪慧芝黃啟清周記源
口試委員(外文):Wei-Yuan ChouHung,Hui-ChihHwang,Chi-ChingChou,Chi-Yuan
口試日期:2011-06-16
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:101
中文關鍵詞:四羥基苯基丙酮酸雙氧合酶
外文關鍵詞:4-hydroxyphenylpyruvate dioxygenase
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四羥基苯基丙酮酸雙氧合酶 (4-HPPD ; 4-Hdyroxyphenylpyruvate dioxygenase)催化4-hydroxyphenylpyruvate(4-HPP)經氧化脫羧、取代基轉移和苯環氧化反應而產生homogentisate,此酶為催化酪胺酸代謝途徑中第二步驟。4-HPPD屬於α-keto acid依賴型氧合酶家族中的一員,也是一種非紫質需鐵氧化酶,其活性中心以2-His 1-carboxylate facial triad的方式與二價鐵離子結合,而主要活性區在C-terminal domain。人類4-HPPD是一個雙聚體的蛋白,單體分子量為44.8kDa,共有393個胺基酸,目前解出的人類4-HPPD晶體結構羧酸端長度只解到羧尾端第384個胺基酸。先前研究指出羧尾端對大鼠4-HPPD活性具有重要性,當羧酸端切除14個胺基酸後會造成4-HPPD酵素活性完全喪失。不同物種經羧酸端序列比對發現不同物種其羧酸端長短不一,雖具有高度保留性,但因羧酸端的高構型變化性,無法提供其在4-HPPD上扮演的角色,因此利用羧酸端切除5(ΔG388)、8(ΔE385)、12(ΔL381) 、13(ΔN380) 、14 (ΔG379) 、15 (ΔR378) 、17 (ΔN376) 、19 (ΔE374)個胺基酸,研究4-HPPD活性及結構影響,以了解羧酸端之功能角色。以圓偏振二色旋光譜、螢光光譜儀和分子篩層析管柱分析蛋白質突變後結構與野生型類似。以HPLC和耗氧測定儀分析活性,發現突變型4-HPPD隨長度切除變短其活性分別為野生型之81%、57%、50%、17%、1% ,而切除15(ΔR378)個胺基酸後幾乎偵測不到活性,由兩種方法分析的結果一致。酵素動力學分析,結果指出突變型4-HPPD羧酸端不同片段切除不影響與其基質之結合親和性(Km值),其kcat及kcat/Km有明顯降低,不同片段切除會影響4-HPPD的催化效率,同時螢光減弱實驗證明羧酸端切除後不影響對基質4-HPP和 Fe2+的結合能力(Kd值)。4-HPPD 利用 DS modeling 模擬人類羧酸端切除胺基酸時會影響羧酸端α-螺旋結構產生位移,進一步影響在羧酸端上的部分胺基酸產生偏移,而造成原本維持結構的氫鍵鍵結消失,因此推測此可能是造成酵素活性下降的原因,總結之,本研究證實人類重組4-HPPD中的羧酸端在其催化功能上扮演著很重要的角色。



4-hydroxypheylpyruvate dioxygenase(4-HPPD) catalyses the second step in tyrosine catabolism involving conversion of 4-hydroxyphenylpyruvate to homogentisate via steps of decarboxylation , substituent migration and aromatic hydroxylation.4-HPPD is a member of the non-heme iron(II)/2-oxoacid-dependent dioxygenase family . In the resolved structure of 4-HPPD , the active site which located in the C-terminal domain and possesses a 2-His-1-carboxylate facial triad for iron binding covered by a C-terminal α-helix . Human 4-HPPD is a homo-dimer with a molecular weight of 44.8 kDa and 393 amino acid for each monomer . Multiple sequence aligment of 4-HPPD had shown a high conservative in C-terminal domain with variable length of the tail . Due to the flexibility , the C-terminal tail in the structure was unresolved . The C-terminus of rat 4-HPPD was reported to be indispensable in enzyme activity,but the detail function of C-terminus in 4-HPPD remains unclear . In the present study , the function of C-terminus in recombinant human 4-HPPD was studied by truncation . Truncated mutants C-terminus of 4-HPPD resulted no effect on the structure as analysed by circular dichroism , fluorescence spectrum and gel filtration chromatography.The activity of ΔG388 , ΔE385 , ΔL381 , ΔN380 and ΔG379 had about 81%、57%、50%、17%、1% activity of wild type 4-HPPD as determined by oxygragh and HPLC assay . When 15 amino acid was truncated from the C-terminus of 4-HPPD , no enzyme activity was detected . Kinetic analysis of mutants 4-HPPD indicated no changes in the Km value but the kcat and kcat/Km were significantly decreased . The Kd values of truncated mutants 4-HPPD were similar to wild type as measured by fluorescence quenching useing Fe2+ or 4-HPP as probe . Simulated mutants structure indicated that truncation in C-terminus of 4-HPPD causes a shift in the C-terminal α-helix leading to movement of R378 and Q375 . The H-binding formed by R378 and E254 , and Q375 and K249 were assums to be important for catalysis . In conclution , this study identified the crucial role of C-terminus in Human 4-HPPD for catalysis .

緒論 ....................................................... 1
實驗材料 ................................................... 8
壹、 藥品試劑 .............................................. 8
貳、 儀器及器材 ............................................ 9
參、 試劑配置 ............................................. 10
實驗方法 .................................................. 14
壹、 構築突變型人類重組 4-HPPD DNA ..................... ..14
一、 設計引子 ................................... .....14
二、 DNA 聚合酶連鎖反應............................. 15
三、 限制酶(restrction enzyme)切目標DNA和載體 ...........17
四、 DNA 膠體電泳 ...................................17
五、 回收 DNA .......................................17
六、 DNA 連結反應 (Ligation) .......................... 18
七、 轉殖 (transformation) .............................. 19
八、 GC Cloning 實驗.................................. 19
九、 勝任細胞 (Competent Cell, DH5α) 的製備 ............ 21
貳、 表現 4-HPPD 蛋白質 .................................. 22
參、 純化 4-HPPD 蛋白質 .................................. 23
肆、 蛋白質含量測定 ....................................... 25
伍、 以 SDS-PAGE 鑑定蛋白質純度和分子量 ..................25
陸、 西方墨點法 (Western blotting) ............................ 26
柒、 圓偏振二色旋分析光譜 (Circular Dichroism) ................27
一、 光譜測定 ........................................ 27
二、 溫度光譜測定..................................... 28
捌、 螢光光譜測定.......................................... 28
一、 蛋白質色胺酸螢光實驗............................. 28
二、 ANS 結合實驗.................................... 29
玖、 螢光減弱法分析 acrylamide 與 4-HPPD 之Ksv 值.......... 29
壹拾、 分子篩層析法 (S-200 column) .........................30
壹拾壹、 耗氧測定儀 (Oxygraph).............................30
壹拾貳、 高效液態層析儀 (HPLC) ........................... 32
壹拾參、 螢光減弱法分析Fe2+、4-HPP與4-HPPD 之結合常數 ....33
壹拾肆、 DS modeling 模擬人類 4-HPPD 結構 ................34
實驗結果 .................................................. 35
壹、 野生型和變異型人類重組 4-HPPD 蛋白質純化 ...........35
一、陰離子交換層析法 (Q Sepharose column) ................35
二、疏水性層析管柱 (Hydrophobic column) ................. 35
三、分子篩層析法 (S-100 column) ......................... 36
四、野生型4-HPPD經純化後,將各步驟純化結果進行SDS-PAGE
分析.............................................36
貳、 野生型與突變型人類4-HPPD純化結果與西方墨點法壓片...36
參、 圓偏振二色旋光譜分析 ............................... 37
肆、 螢光實驗分析 ....................................... 37
伍、 ANS結合分析........................................ 38
陸、 4-HPPD螢光減弱實驗分析............................. 38
柒、 分子篩層析法 (S-200 column) .......................... 39
捌、 利用圓偏振二色旋光分析圖變蛋白質之熱安定性 ......... 39
玖、 野生型及突變型 4-HPPD 活性分析 .................... 39
一、 耗氧測定儀 ...................................... 39
二、 利用高效液態層析儀 (HPLC) 分析反應產物 HG (以不同濃
度4-HPP基質分析) .................................40
三、 利用高效液態層析儀(HPLC)分析不同濃度Fe2+離子影響下動 力學分析 .......................................42
壹拾、 螢光減弱分析螢光減弱分析4-HPPD與Fe2+及4-HPP之Kd值.42
壹拾壹、 突變型4-HPPD 結構模擬分析.......................43
討論 ...................................................... 44
參考文獻 .................................................. 82

1.Hayaishi, O., and Nozaki, M. (1969) Nature and mechanisms of oxygenases, Science 164, 389-396.
2.Yu, B., Edstrom, W. C., Benach, J., Hamuro, Y., Weber, P. C., Gibney, B. R., and Hunt, J. F. (2006) Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB, Nature 439, 879-884.
3.Mellor, A. L., and Munn, D. H. (2004) IDO expression by dendritic cells: tolerance and tryptophan catabolism, Nat Rev Immunol 4, 762-774.
4.Neve, S., Aarenstrup, L., Tornehave, D., Rahbek-Nielsen, H., Corydon, T. J., Roepstorff, P., and Kristiansen, K. (2003) Tissue distribution, intracellular localization and proteolytic processing of rat 4-hydroxyphenylpyruvate dioxygenase, Cell Biol Int 27, 611-624.
5.Lindblad, B., Lindstedt, G., Lindstedt, S., and Rundgren, M. (1977) Purification and some properties of human 4-hydroxyphenylpyruvate dioxygenase (I), J Biol Chem 252, 5073-5084.
6.Moran, G. R. (2005) 4-Hydroxyphenylpyruvate dioxygenase, Arch Biochem Biophys 433, 117-128.
7.Endo, F., Awata, H., Tanoue, A., Ishiguro, M., Eda, Y., Titani, K., and Matsuda, I. (1992) Primary structure deduced from complementary DNA sequence and expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase. Evidence that the enzyme is a homodimer of identical subunits homologous to rat liver-specific alloantigen F, J Biol Chem 267, 24235-24240.
8.Hegg, E. L., and Que, L., Jr. (1997) The 2-His-1-carboxylate facial triad--an emerging structural motif in mononuclear non-heme iron(II) enzymes, Eur J Biochem 250, 625-629.
9.Pascal, R. A., Jr., Oliver, M. A., and Chen, Y. C. (1985) Alternate substrates and inhibitors of bacterial 4-hydroxyphenylpyruvate dioxygenase, Biochemistry 24, 3158-3165.
10.Johnson-Winters, K., Purpero, V. M., Kavana, M., Nelson, T., and Moran, G. R. (2003) (4-Hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis: the basis for ordered substrate addition, Biochemistry 42, 2072-2080.
11.Falk, J., Andersen, G., Kernebeck, B., and Krupinska, K. (2003) Constitutive overexpression of barley 4-hydroxyphenylpyruvate dioxygenase in tobacco results in elevation of the vitamin E content in seeds but not in leaves, FEBS Lett 540, 35-40.
12.Garcia, I., Rodgers, M., Pepin, R., Hsieh, T. F., and Matringe, M. (1999) Characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from Arabidopsis in transgenic tobacco, Plant Physiol 119, 1507-1516.
13.Romagni, J. G., Meazza, G., Nanayakkara, N. P., and Dayan, F. E. (2000) The phytotoxic lichen metabolite, usnic acid, is a potent inhibitor of plant p-hydroxyphenylpyruvate dioxygenase, FEBS Lett 480, 301-305.
14.Meazza, G., Scheffler, B. E., Tellez, M. R., Rimando, A. M., Romagni, J. G., Duke, S. O., Nanayakkara, D., Khan, I. A., Abourashed, E. A., and Dayan, F. E. (2002) The inhibitory activity of natural products on plant p-hydroxyphenylpyruvate dioxygenase, Phytochemistry 60, 281-288.
15.Yang, C., Pflugrath, J. W., Camper, D. L., Foster, M. L., Pernich, D. J., and Walsh, T. A. (2004) Structural basis for herbicidal inhibitor selectivity revealed by comparison of crystal structures of plant and mammalian 4-hydroxyphenylpyruvate dioxygenases, Biochemistry 43, 10414-10423.
16.Kavana, M., and Moran, G. R. (2003) Interaction of (4-hydroxyphenyl)pyruvate dioxygenase with the specific inhibitor 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione, Biochemistry 42, 10238-10245.
17.Brownlee, J. M., Johnson-Winters, K., Harrison, D. H., and Moran, G. R. (2004) Structure of the ferrous form of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis in complex with the therapeutic herbicide, NTBC, Biochemistry 43, 6370-6377.
18.Aarenstrup, L., Falch, A. M., Jakobsen, K. K., Neve, S., Henriksen, L. L., Tommerup, N., Leffers, H., and Kristiansen, K. (2002) Expression and post-translational modification of human 4-hydroxy-phenylpyruvate dioxygenase, Cell Biol Int 26, 615-625.
19.Lindstedt, S., Holme, E., Lock, E. A., Hjalmarson, O., and Strandvik, B. (1992) Treatment of hereditary tyrosinaemia type I by inhibition of 4-hydroxyphenylpyruvate dioxygenase, Lancet 340, 813-817.
20.Ruetschi, U., Cerone, R., Perez-Cerda, C., Schiaffino, M. C., Standing, S., Ugarte, M., and Holme, E. (2000) Mutations in the 4-hydroxyphenylpyruvate dioxygenase gene (HPD) in patients with tyrosinemia type III, Hum Genet 106, 654-662.
21.Gunsior, M., Ravel, J., Challis, G. L., and Townsend, C. A. (2004) Engineering p-hydroxyphenylpyruvate dioxygenase to a p-hydroxymandelate synthase and evidence for the proposed benzene oxide intermediate in homogentisate formation, Biochemistry 43, 663-674.
22.Wada, G. H., Fellman, J. H., Fujita, T. S., and Roth, E. S. (1975) Purification and properties of avian liver p-hydroxyphenylpyruvate hydroxylase, J Biol Chem 250, 6720-6726.
23.Serre, L., Sailland, A., Sy, D., Boudec, P., Rolland, A., Pebay-Peyroula, E., and Cohen-Addad, C. (1999) Crystal structure of Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway, Structure 7, 977-988.
24.Fritze, I. M., Linden, L., Freigang, J., Auerbach, G., Huber, R., and Steinbacher, S. (2004) The crystal structures of Zea mays and Arabidopsis 4-hydroxyphenylpyruvate dioxygenase, Plant Physiol 134, 1388-1400.
25.Han, S., Eltis, L. D., Timmis, K. N., Muchmore, S. W., and Bolin, J. T. (1995) Crystal structure of the biphenyl-cleaving extradiol dioxygenase from a PCB-degrading pseudomonad, Science 270, 976-980.
26.Hogan, D. A., Smith, S. R., Saari, E. A., McCracken, J., and Hausinger, R. P. (2000) Site-directed mutagenesis of 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate dioxygenase. Identification of residues involved in metallocenter formation and substrate binding, J Biol Chem 275, 12400-12409.
27.Rundgren, M. (1977) Multiple forms of human 4-hydroxyphenylpyruvate dioxygenase (II), J Biol Chem 252, 5085-5093.
28.Lee, M. H., Zhang, Z. H., MacKinnon, C. H., Baldwin, J. E., and Crouch, N. P. (1996) The C-terminal of rat 4-hydroxyphenylpyruvate dioxygenase is indispensable for enzyme activity, FEBS Lett 393, 269-272.
29.Hiratsuka, T. (1983) Acrylamide fluorescence quenching studies on the actin-induced change in protein dynamics in the subfragment-1/subfragment-2 link region of cardiac myosin, J Biochem 93, 875-882.
30.Dunning Hotopp, J. C., Auchtung, T. A., Hogan, D. A., and Hausinger, R. P. (2003) Intrinsic tryptophan fluorescence as a probe of metal and alpha-ketoglutarate binding to TfdA, a mononuclear non-heme iron dioxygenase, J Inorg Biochem 93, 66-70.
31.Raspail, C., Graindorge, M., Moreau, Y., Crouzy, S., Lefebvre, B., Robin, A. Y., Dumas, R., and Matringe, M. (2011) 4-hydroxyphenylpyruvate dioxygenase catalysis: identification of catalytic residues and production of a hydroxylated intermediate shared with a structurally unrelated enzyme, J Biol Chem.



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