跳到主要內容

臺灣博碩士論文加值系統

(18.97.9.169) 您好!臺灣時間:2025/02/16 06:24
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:鄒穎佳
研究生(外文):Yin-Chia Chou
論文名稱:紫茉莉中參與甜菜⾊素⽣合成之4,5-多巴雙加氧酶的結構與功能探討
論文名稱(外文):Functional and structural studies of a 4,5-DOPA dioxygenase involved in betalain pigment biosynthesis from Mirabilis jalapa
指導教授:徐駿森
指導教授(外文):Chun-Hua Hsu
口試日期:2017-07-04
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:90
中文關鍵詞:甜菜⾊素甜菜醛氨酸外雙加氧酶紫茉莉植物⾊素晶體結構
外文關鍵詞:Betalainsbetalamic acidextradiol dioxygenaseMairabilis jalapaplant pigmentcrystal structure
相關次數:
  • 被引用被引用:0
  • 點閱點閱:349
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
甜菜色素是一含氮的水溶性色素,存在於石竹目(Caryophyllales) 植物的花、果實或其他部位。甜菜醛胺酸是構成天然甜菜色素的基本單元,以左多巴 (3,4-dihydroxy-L-phenylalanine , L-DOPA) 作為前驅物,由關鍵酵素4,5-多巴雙加氧酶 (4,5-DOPA-extradiol dioxygenase) 進行催化生成中間產物 4,5-seco-dopa,在經過自發性分子內環化最後生成甜菜醛胺酸。本論文對紫茉莉 (Mairabilis jalapa) 的4,5-多巴雙加氧酶 (簡稱mjDOD) 進行相關功能與結構特性探討,圓二色光譜儀顯示mjDOD具有良好的熱穩定性,Tm約為 58°C,在pH 3.5 ~ pH 8.5的環境中都可以維持正確折疊,而其活性最適pH值為6.0。此外,mjDOD之晶體結構亦被解構至解析度為2.66 Å,結構顯示mjDOD 之催化中心由 H9、H47 和 H222 與二價鐵離子形成配位,推測此酵素可利用非血基質鐵 (non-heme Fe(II)) 催化開環反應,因其催化中心結構特徵與其他外雙加氧酶相似。在催化中心的附近有兩個具有保守性的胺基酸 H112 和 H168 ,對其做點突變後會使酵素完全失去活性,表示這兩個胺基酸在催化反應中扮演重要角色。根據這些結果,我們提出 L-DOPA 與 mjDOD 結合的可能模式與酵素催化機制。據我們所知,此mjDOD的晶體結構為第一個被解析的植物類型4,5-多巴雙加氧酶結構。
Betalains are water-soluble nitrogenous pigments, synthesized in flowers, fruits and other tissues of the plant order Caryophyllales. Betalamic acid is the structural unit of natural pigments betalains. The formation of betalamic acid from the precursor amino acid L-DOPA (3,4-dihydroxy-L-phenylalanine) is catalyzed by the key enzyme 4,5-DOPA-dioxygenase followed by intramolecular cyclization of the 4,5-seco-dopa intermediate. In this thesis, we examined the functional properties and structural characteristics of a 4,5-DOPA-dioxygenase (mjDOD) from Mairabilis jalapa. Circular dichroism spectra showed that mjDOD has good thermal stability with the Tm value as 58°C, and can maintain a correct fold at pH range of 3.5 to 8.5. The pH optimum for enzyme activity toward L-DOPA was shown to pH 6.0. In addition, the crystal structure of mjDOD was determined at 2.66 Å resolution, showing that the Fe-ligand residues are H9, H47 and H222 and indicating that the enzyme uses non-heme Fe(II) to catalyzing a ring opening reaction, which also found in many other extradiol dioxygenase. Since two conserved residues H112 and H168 are observed near the active site, site-directed mutagenesis was conducted for further examination. Interestingly, the mutants are totally inactive. According to these results, the substrate binding mode and catalytic mechanism of mjDOD are proposed. To the best of our knowledge, the crystal structure of mjDOD is the first solved plant 4,5-DOPA-dioxygenase.
目錄
壹、前言 1
一、 植物色素簡介 1
二、 甜菜色素 (Betalains) 1
三、 甜菜醛胺酸的合成 (formation of betalamic acid) 2
四、 甜菜色素的合成 (formation of betalains) 3
五、 雙加氧酶 (Dioxygenase) 6
六、 4,5-多巴雙加氧酶(4,5-DOPA-extradiol-dioxygenase) 8
研究目的 12
貳、材料與方法 13
一、 實驗材料 13
1. mjDOD 基因 13
2. 菌株及培養系統 13
二、 實驗方法 13
1. 蛋白質表現載體之構築 13
2. 大腸桿菌勝任細胞 (Competent cell) 之製備 15
3. 轉形作用 (Transformation) 15
4. 抽取質體 DNA (Plasmid DNA) 15
5. DNA 定序與胺基酸序列比對 16
6. SDS 蛋白質膠體電泳分析 (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) 16
7. 重組蛋白質之表現 17
8. 重組蛋白質之純化 17
i. 鎳離子親和性樹脂管柱 18
ii. 離子交換樹脂管住 18
9. 蛋白質透析與濃縮 19
10. 蛋白質濃度測定 19
11. 膠體過濾層析法 (Gel Filtration Chromatography) 20
12. 酵素活性測試 21
13. mjDOD 之最適pH值 21
14. mjDOD 之最適溫度 22
15. 不同金屬離子存在下之mjDOD活性 22
16. 酵素動力學 22
17. 圓二色光譜儀 23
18. 示差掃描螢光蛋白質熱穩定性測試 24
19. X-ray 晶體繞射技術解析蛋白質結構 25
i. 蛋白質結晶測試 25
ii. 蛋白質晶體條件篩選 25
iii. 蛋白質晶體形成條件微調 26
iv. 適合抗凍劑之篩選 26
v. X-ray晶體繞射數據收集及處理 27
vi. 蛋白質晶體結構建立 28
vii. Ramachandran plot 28
20. 蛋白質結構比對 29
21. 定點突變實驗 29
參、研究結果 31
一、 製作表現載體並讓 mjDOD 之 C 端帶有 His-tag 31
二、 小量表現蛋白質以尋找適合之蛋白質誘導條件 31
三、 mjDOD 大量表現的純化 32
四、 以膠體過濾層析法分析 mjDOD 的分子量 33
五、 pH值對 mjDOD 之影響 33
六、 溫度對 mjDOD 之影響 34
七、 酵素動力學 36
八、 不同金屬離子對酵素活性的影響 36
九、 蛋白質晶體培養 37
十、 晶體抗凍既篩選 38
十一、 X-ray 繞射數據分析 38
十二、 蛋白質結晶結構建立 38
十三、 mjDOD 的蛋白質結構 39
1. 不同雙加氧酶之結構比較 39
2. mjDOD 結構之催化中心中有一分子的檸檬酸 40
十四、 以檸檬酸為抑制劑之酵素動力學測定 41
十五、 預測L-DOPA之結合位置 42
十六、 選擇定點突變的胺基酸位置 43
十七、 H112F與 H168F突變株之酵素活性 43
肆、討論 45
一、 H112與H168在 mjDOD中所扮演的角色 45
二、 推測mjDOD 酵素反應機制 45
三、 比較mjDOD和YgiD於L-DOPA結合位之結構差異 46
四、 比較不同4,5-DOPA-extradiol-dioxygenase 47
伍、結論 49
陸、圖表 50
柒、參考文獻 79
附錄 85
Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66 (Pt 2):213-221.
Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD (2012) Towards automated crystallographic structure refinement with phenix.refine. cta Crystallogr D Biol Crystallogr 68 (Pt 4):352-367.
Afonine PV, Moriarty NW, Mustyakimov M, Sobolev OV, Terwilliger TC, Turk D, Urzhumtsev A, Adams PD (2015) FEM: feature-enhanced map. Acta Crystallogr D Biol Crystallogr 71 (Pt 3):646-666.
Babos M, Halasz K, Zagyva T, Zold-Balogh A, Szego D, Bratek Z (2011) Preliminary notes on dual relevance of ITS sequences and pigments in Hygrocybe taxonomy. Persoonia 26:99-107.
Brockington SF, Walker RH, Glover BJ, Soltis PS, Soltis DE (2011) Complex pigment evolution in the Caryophyllales. New Phytol 190 (4):854-864
Canovas FG, Garcia-Carmona F, Sanchez JV, Pastor JL, Teruel JA (1982) The role of pH in the melanin biosynthesis pathway. J Biol Chem 257 (15):8738-8744
Christinet L, Burdet FRX, Zaiko M, Hinz U, Zryd JP (2004) Characterization and functional identification of a novel plant 4,5-extradiol dioxygenase involved in betalain pigment biosynthesis in Portulaca grandiflora. Plant Physiol 134 (1):265-274.
Dunwell JM, Culham A, Carter CE, Sosa-Aguirre CR, Goodenough PW (2001) Evolution of functional diversity in the cupin superfamily. Trends in Biochem Sci 26 (12):740-746.
Eltis LD, Bolin JT (1996) Evolutionary relationships among extradiol dioxygenases. J Bacteriol 178 (20):5930-5937.
Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60 (Pt 12 Pt 1):2126-2132.
Gandia-Herrero F (2005) Betaxanthins as Substrates for Tyrosinase. An Approach to the Role of Tyrosinase in the Biosynthetic Pathway of Betalains. Plant Physiol 138 (1):421-432.
Gandía-Herrero F, Escribano J, García-Carmona F (2007) Characterization of the Activity of Tyrosinase on Betanidin. J Agric Food Chem 55 (4):1546-1551.
Gandia-Herrero F, Escribano J, Garcia-Carmona F (2010) Structural implications on color, fluorescence, and antiradical activity in betalains. Planta 232 (2):449-460.
Gandia-Herrero F, Escribano J, Garcia-Carmona F (2012) Purification and antiradical properties of the structural unit of betalains. J Nat Prod 75 (6):1030-1036.
Gandia-Herrero F, Garcia-Carmona F (2012) Characterization of recombinant Beta vulgaris 4,5-DOPA-extradiol-dioxygenase active in the biosynthesis of betalains. Planta 236 (1):91-100.
Gandia-Herrero F, Garcia-Carmona F (2013) Biosynthesis of betalains: yellow and violet plant pigments. Trends Plant Sci 18 (6):334-343.
Gandia-Herrero F, Garcia-Carmona F (2014) Escherichia coli protein YgiD produces the structural unit of plant pigments betalains: characterization of a prokaryotic enzyme with DOPA-extradiol-dioxygenase activity. Appl Microbiol Biotechnol 98 (3):1165-1174.
Gandia-Herrero F, Garcia-Carmona F, Escribano J (2005) Botany: floral fluorescence effect. Nature 437 (7057):334.
Girod P-A, Zryd J-P (1991) Biogenesis of betalains: Purification and partial characterization of dopa 4,5-dioxygenase from Amanita muscaria. Phytochemistry 30 (1):169-174.
Godoy-Alcántar C, Yatsimirsky AK, Lehn JM (2005) Structure-stability correlations for imine formation in aqueous solution. J Phys Org Chem 18 (10):979-985.
Green BR, Durnford DG (1996) The Chlorophyll-Carotenoid Proteins of Oxygenic Photosynthesis. Annu Rev Plant Physiol Plant Mol Biol 47:685-714.
Groce SL, Lipscomb JD (2003) Conversion of extradiol aromatic ring-cleaving homoprotocatechuate 2,3-dioxygenase into an intradiol cleaving enzyme. J Am Chem Soc 125 (39):11780-11781.
Guengerich FP (2001) Common and Uncommon Cytochrome P450 Reactions Related to Metabolism and Chemical Toxicity. Chem Res Toxicol 14 (6):611-650.
Hans J, Brandt W, Vogt T (2004) Site-directed mutagenesis and protein 3D-homology modelling suggest a catalytic mechanism for UDP-glucose-dependent betanidin 5-O-glucosyltransferase from Dorotheanthus bellidiformis. Plant J 39 (3):319-333.
Hatlestad GJ, Sunnadeniya RM, Akhavan NA, Gonzalez A, Goldman IL, McGrath JM, Lloyd AM (2012) The beet R locus encodes a new cytochrome P450 required for red betalain production. Nat Genet 44 (7):816-820.
Hinz UG, Fivaz J, Girod PA, Zyrd JP (1997) The gene coding for the DOPA dioxygenase involved in betalain biosynthesis in Amanita muscaria and its regulation. Mol Gen Genet 256 (1):1-6
Holm L, Rosenstrom P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38 (Web Server issue):W545-549.
Jouanneau Y (2010) Oxidative Inactivation of Ring-Cleavage Extradiol Dioxygenases: Mechanism and Ferredoxin-Mediated Reactivation.1071-1079.
Kishima Y, Suiko M, Adachi T (1991) Betalain Pigmentation in Petal of Portulaca is Preceded by a Dramatic Tyrosine Accumulation. J Plant Physiol 137 (4):505-506.
Koehntop KD, Emerson JP, Que L, Jr. (2005) The 2-His-1-carboxylate facial triad: a versatile platform for dioxygen activation by mononuclear non-heme iron(II) enzymes. J Biol Inorg Chem 10 (2):87-93.
Kovaleva EG, Lipscomb JD (2007) Crystal structures of Fe2+ dioxygenase superoxo, alkylperoxo, and bound product intermediates. Science 316 (5823):453-457.
Land EJ, Riley PA (2000) Spontaneous Redox Reactions of Dopaquinone and the Balance between the Eumelanic and Phaeomelanic Pathways. Pigment Cell Res 13 (4):273-277.
Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8 (4):477-486
Lechner JF, Wang LS, Rocha CM, Larue B, Henry C, McIntyre CM, Riedl KM, Schwartz SJ, Stoner GD (2010) Drinking water with red beetroot food color antagonizes esophageal carcinogenesis in N-nitrosomethylbenzylamine-treated rats. J Med Food 13 (3):733-739.
Li J, Christensen BM (1993) Identification of Products and Intermediates During L-Dopa Oxidation to Dopachrome Using High Pressure Liquid Chromatography with Electrochemical Detection. J Liq Chromatogr 16 (5):1117-1133.
Lin G, Reid G, Bugg TD (2001) Extradiol oxidative cleavage of catechols by ferrous and ferric complexes of 1,4,7-triazacyclononane: insight into the mechanism of the extradiol catechol dioxygenases. J Am Chem Soc 123 (21):5030-5039
Lu X, Wang Y, Zhang Z (2009) Radioprotective activity of betalains from red beets in mice exposed to gamma irradiation. Eur J Pharmacol 615 (1-3):223-227.
McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40 (Pt 4):658-674.
Mueller LA, Hinz U, Zryd JP (1997) The formation of betalamic acid and muscaflavin by recombinant dopa-dioxygenase from Amanita. Phytochemistry 44 (4):567-569.
Nambara E, Marion-Poll A (2005) Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol 56:165-185.
Niyogi KK (2000) Safety valves for photosynthesis. Curr Opin Plant Biol 3 (6):455-460
Riley PA (2000) Tyrosinase Kinetics: A Semi-quantitative Model of the Mechanism of Oxidation of Monohydric and Dihydric Phenolic Substrates. J Theor Biol 203 (1):1-12.
Sasaki N (2005) Isolation and Characterization of cDNAs Encoding an Enzyme with Glucosyltransferase Activity for cyclo-DOPA from Four O''clocks and Feather Cockscombs. Plant Cell Physiol 46 (4):666-670.
Sasaki N, Abe Y, Goda Y, Adachi T, Kasahara K, Ozeki Y (2009) Detection of DOPA 4,5-dioxygenase (DOD) activity using recombinant protein prepared from Escherichia coli cells harboring cDNA encoding DOD from Mirabilis jalapa. Plant Cell Physiol 50 (5):1012-1016.
Schliemann W, Kobayashi N, Strack D (1999) The Decisive Step in Betaxanthin Biosynthesis Is a Spontaneous Reaction1. Plant Physiol 119 (4):1217-1232.
Schliemann W, Steiner U, Strack D (1998) Betanidin formation from dihydroxyphenylalanine in a model assay system. Phytochemistry 49 (6):1593-1598
Sekiguchi H, Ozeki Y, Sasaki N (2010) In vitro synthesis of betaxanthins using recombinant DOPA 4,5-dioxygenase and evaluation of their radical-scavenging activities. J Agric Food Chem 58 (23):12504-12509.
Shimada S, Otsuki H, Sakuta M (2007) Transcriptional control of anthocyanin biosynthetic genes in the Caryophyllales. J Exp Bot 58 (5):957-967.
Shu L, Chiou YM, Orville AM, Miller MA, Lipscomb JD, Que L, Jr. (1995) X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase. Implications for the extradiol cleavage mechanism. Biochemistry 34 (20):6649-6659
Sreekanth D, Arunasree MK, Roy KR, Chandramohan Reddy T, Reddy GV, Reddanna P (2007) Betanin a betacyanin pigment purified from fruits of Opuntia ficus-indica induces apoptosis in human chronic myeloid leukemia Cell line-K562. Phytomedicine 14 (11):739-746.
Stintzing F, Schliemann W (2007) Pigments of fly agaric (Amanita muscaria). Z Naturforsch C 62 (11-12):779-785
Stintzing FC, Schieber A, Carle R (2002) Betacyanins in fruits from red-purple pitaya, Hylocereus polyrhizus (Weber) Britton & Rose. Food Chem 77 (1):101-106.
Strack D, Vogt T, Schliemann W (2003) Recent advances in betalain research. Phytochemistry 62 (3):247-269.
Sugimoto K, Senda M, Kasai D, Fukuda M, Masai E, Senda T (2014) Molecular mechanism of strict substrate specificity of an extradiol dioxygenase, DesB, derived from Sphingobium sp. SYK-6. PLoS One 9 (3):e92249.
Sugimoto K, Senda T, Aoshima H, Masai E, Fukuda M, Mitsui Y (1999) Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions. Structure 7 (8):953-965
Tanaka Y, Sasaki N, Ohmiya A (2008) Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J 54 (4):733-749.
Toivonen PMA, Brummell DA (2008) Biochemical bases of appearance and texture changes in fresh-cut fruit and vegetables. Postharvest Biol Technol 48 (1):1-14.
Trezzini GF, Zrÿb JP (1991) Characterization of some natural and semi-synthetic betaxanthins. Phytochemistry 30 (6):1901-1903.
Vaillancourt FH, Barbosa CJ, Spiro TG, Bolin JT, Blades MW, Turner RF, Eltis LD (2002) Definitive evidence for monoanionic binding of 2,3-dihydroxybiphenyl to 2,3-dihydroxybiphenyl 1,2-dioxygenase from UV resonance Raman spectroscopy, UV/Vis absorption spectroscopy, and crystallography. J Am Chem Soc 124 (11):2485-2496
Vaillancourt FH, Bolin JT, Eltis LD (2006) The ins and outs of ring-cleaving dioxygenases. Crit Rev Biochem Mol Biol 41 (4):241-267.
Wyler H, Chiovini J (1968) Die Synthese von Cyclodopa (Leukodopachrom). Helvetica Chimica Acta 51 (6):1476-1494.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top