博碩士論文 102224002 詳細資訊




以作者查詢圖書館館藏 以作者查詢臺灣博碩士 以作者查詢全國書目 勘誤回報 、線上人數:28 、訪客IP:3.236.16.13
姓名 曾昱尊(Yu-Tsuen Tsen)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 古生菌嗜酸熱硫化葉菌的乙醯乳酸還原異構酶的晶體結構以及穩定性
(Crystal structure and stability of acetohydroxyacid isomeroreductase from hyperthermophilic Sulfolobus acidocaldarius)
相關論文
★ 硫化屬古生菌中的酮醇酸還原異構酶結構分析★ 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
★ 嗜酸熱硫化葉菌的DNA結合蛋白Saci_0101之結構與功能分析★ PDCD5蛋白在Sulfolobus solfataricus 古生菌的結構與功能分析
★ 嗜酸熱硫化葉菌中去氧核醣核酸結合蛋白Saci_1212之結構性及功能性分析★ 硫磺礦硫化葉菌程序性細胞死亡蛋白5晶體結構分析及其與DNA的相互作用
★ 嗜酸熱硫化葉菌中DNA結合蛋白Sac10b之結構分析及其與DNA相互作用★ 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
★ 脂肪酸特異互養棲熱菌酮醇酸還原異構酶之晶體結構及活性分析
檔案 [Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   至系統瀏覽論文 ( 永不開放)
摘要(中) 酵素acetohydroxy acid isomeroreductase (AHIR, EC 1.1.1.86)是在細菌、植物和真菌支鏈型胺基酸合成的第二個酵素,且不會出現在動物細胞中。利用這項動物與其他物種間的差異,可以將酵素AHIR當作抗菌劑或是除草劑的抑制目標,酵素AHIR是再生合成過程中涉及烷基移動以及還原的兩步反應,並且需要輔因子Mg2+以及NAD(P)H的參與。我們同樣在古生菌嗜酸熱硫化葉菌 (Sulfolobus acidocaldarius)中發現這個酵素(Sac_AHIR),此古生菌是在黃石公園的火山口溫泉發現的,屬古泉菌門。我們利用大腸桿菌當宿主細胞,大量表達Sac_AHIR蛋白質,純化並養出蛋白質晶體,以X光繞射收到最高解析度為1.75Å的繞射數據,並用有Se-Met標定的AHIR進行多波長異常散射實驗解出晶體相位,建立蛋白質模型。每個AHIR在自然狀態下形成雙聚體結構,而每個蛋白質在序列上分成兩個部分,N端的Rossmann domain以及C端的knotted domain。兩個knotted domain可以相互作用幫助雙聚體組合,並且形成兩個有功能的活性位。經由比對發現Sac_AHIR的Rossmann domain上的特殊序列β2αB-loop是屬於7個胺基酸類型且偏向親合NADPH作為輔因子。利用圓二色光譜研究可以發現,Sac_AHIR在65°C時會稍微變性直至95°C時會完全變性,而蛋白質的二級結構在pH 3到pH 8之間並沒有顯著的差異。我們推測即使Sac_AHIR在75°C和pH 2的極端溫泉環境,此蛋白質酵素在細胞中依然能保持活性。Sac_AHIR可在高溫與不同酸鹼程度的環境下,保持蛋白質折疊正確,性質相較細菌或植物內的AHIR來得穩定,在生物科技或生質能源上更有應用的潛力
摘要(英) Acetohydroxyacid isomeroreductase (AHIR) is the second key enzyme involved in the branched-chain amino acid biosynthetic pathways which is found in bacteria, fungi and plants but not in animals. This difference in metabolism between animals and microorganisms makes AHIR an attractive target for the development of specific herbicides and antimicrobial agents. Moreover, it is a bifunctional enzyme that catalyzes two reactions, alkyl migration and reduction, and requires Mg2+ and NAD(P)H for activity. Here we present the crystal structure at 1.75 Å resolution of the Sac_AHIR of Sulfolobus acidocaldarius. Sac_AHIR exists as homodimer in solution and each monomer is composed of two types of domains, an N-terminal Rossmann domain and a C-terminal knotted domains. Two intertwined knotted domains are required for formation of the homodimer and two AHIR active sites. Analysis of the amino acid sequences of the Rossmann fold’s β2αB-loop displays that Sac_AHIR has a seven-residue loop, LEREGNS, which prefers using NADPH as the cofactor. Circular dichroism spectrometric analysis showed that Sac_AHIR was denatures slightly at 65°C but unfold completely at 95 °C. The CD spectra of Sac_AHIR was almost identical between at the range of pH 3.0 to 8.0 at 25 °C. The CD results revealed that Sac_AHIR is thermal stable and acid tolerant. Our study suggests that Sac_AHIR might retain activity under the extreme growth environments at temperature 75 °C and pH 2 in solfataric springs.
關鍵字(中) ★ 晶體繞射
★ 嗜酸熱硫化葉菌
★ 乙醯乳酸還原異構酶
關鍵字(英) ★ Crystal structure
★ Sulfolobus acidocaldarius
★ acetohydroxyacid isomeroreductase
論文目次 目 錄
摘要--------------------------------------------ii
Abstract----------------------------------------iv
誌謝--------------------------------------------v
目錄--------------------------------------------vi
介紹--------------------------------------------1
1-1菌種-----------------------------------------1
1-1-1古生菌(Archaea)----------------------------1
1-1-2 嗜酸熱硫化葉菌(Sulfolobus acidocaldarius)--1
1-1-3 Sulfolobus acidocaldarius特性-------------2
1-2 酵素 AHIR-----------------------------------3
1-2-1 支鏈型胺基酸(BCAA)的生合成------------------3
1-2-2 AHIR的結構和分類----------------------------5
1-2-3 抑制物及其應用------------------------------10
1-2-4 生質能源上的應用----------------------------10
1-3研究動機--------------------------------------12
2.方法與材料-------------------------------------13
2-1晶體形成--------------------------------------13
2-2布拉格定律(Bragg’s low)-----------------------14
2-3蛋白質繞射------------------------------------15
2-4相位問題--------------------------------------17
2-5蛋白質的圓二色光性質---------------------------19
2-6材料------------------------------------------20
2-6-1分生----------------------------------------20
3.實驗結果---------------------------------------22
3-1基因提取、轉殖 ---------------------------------22
3-2蛋白質表現測試---------------------------------23
3-3蛋白質大量表現 ---------------------------------24
3-4耐熱測試---------------------------------------25
3-5純化步驟---------------------------------------26
3-7蛋白質長晶-------------------------------------28
3-9序列比對---------------------------------------30
3-10結構描述--------------------------------------32
4附錄--------------------------------------------38
4-1晶體繞射操作-----------------------------------38
4-2 CCp4i----------------------------------------38
4-2-1檔案轉換------------------------------------38
4-2-2合併.mtz檔案--------------------------------39
4-2-3找重原子位置 ---------------------------------39
4-2-4計算結構相位 ---------------------------------39
4-2-5增強電子密度圖Density Modification-----------40
4-2-6建立R-free data-----------------------------40
4-2-7結構建立------------------------------------40
4-2-8用 Molecular Replace建立蛋白質結構 (MR)------41
4-3用pymol外掛程式APBS Tools來產生表面電荷分布圖---41
4-4 Bioeditor-----------------------------------42
附錄 圓二色光溫度測試----------------------------43
參考文獻-----------------------------------------59
參考文獻 H. F. Sturt, R. E. Summons, K. Smith, M. Elvert, K. U. Hinrichs, Intact polar membrane lipids in prokaryotes and sediments deciphered by high-performance liquid chromatography/electrospray ionization multistage mass spectrometry--new biomarkers for biogeochemistry and microbial ecology. Rapid Commun Mass Spectrom 18, 617-628 (2004).
2. L. Chen et al., The genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota. J Bacteriol 187, 4992-4999 (2005).
3. S. Berkner, D. Grogan, S. V. Albers, G. Lipps, Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea. Nucleic Acids Res 35, e88 (2007).
4. S. K. Chunduru, G. T. Mrachko, K. C. Calvo, Mechanism of ketol acid reductoisomerase--steady-state analysis and metal ion requirement. Biochemistry 28, 486-493 (1989).
5. K. Thomazeau et al., Structure of spinach acetohydroxyacid isomeroreductase complexed with its reaction product dihydroxymethylvalerate, manganese and (phospho)-ADP-ribose. Acta Crystallogr D Biol Crystallogr 56, 389-397 (2000).
6. S. Brinkmann-Chen, J. K. Cahn, F. H. Arnold, Uncovering rare NADH-preferring ketol-acid reductoisomerases. Metab Eng 26, 17-22 (2014).
7. R. Dumas, V. Biou, F. Halgand, R. Douce, R. G. Duggleby, Enzymology, structure, and dynamics of acetohydroxy acid isomeroreductase. Acc Chem Res 34, 399-408 (2001).
8. R. Dumas, M. C. Butikofer, D. Job, R. Douce, Evidence for two catalytically different magnesium-binding sites in acetohydroxy acid isomeroreductase by site-directed mutagenesis. Biochemistry 34, 6026-6036 (1995).
9. S. Atsumi, T. Hanai, J. C. Liao, Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86-89 (2008).
10. F. Halgand et al., Characterization of the conformational changes of acetohydroxy acid isomeroreductase induced by the binding of Mg2+ ions, NADPH, and a competitive inhibitor. Biochemistry 38, 6025-6034 (1999).
11. H. J. Ahn et al., Crystal structure of class I acetohydroxy acid isomeroreductase from Pseudomonas aeruginosa. J Mol Biol 328, 505-515 (2003).
12. W. R. Taylor, A deeply knotted protein structure and how it might fold. Nature 406, 916-919 (2000).
13. S. Bastian et al., Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli. Metab Eng 13, 345-352 (2011).
14. S. Brinkmann-Chen et al., General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH. Proc Natl Acad Sci U S A 110, 10946-10951 (2013).
15. R. Dumas et al., Isolation and kinetic properties of acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) chloroplasts overexpressed in Escherichia coli. Biochem J 288 ( Pt 3), 865-874 (1992).
16. R. Dumas et al., Interactions of plant acetohydroxy acid isomeroreductase with reaction intermediate analogues: correlation of the slow, competitive, inhibition kinetics of enzyme activity and herbicidal effects. Biochem J 301 ( Pt 3), 813-820 (1994).
17. S. Epelbaum, D. M. Chipman, Z. Barak, Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium. J Bacteriol 178, 1187-1196 (1996).
18. X. H. Liu et al., Synthesis, bioactivity, theoretical and molecular docking study of 1-cyano-N-substituted-cyclopropanecarboxamide as ketol-acid reductoisomerase inhibitor. Bioorg Med Chem Lett 17, 3784-3788 (2007).
19. W. H. Bragg, The Analysis of Crystal Structure by X-Rays. Science 60, 139-149 (1924).
20. S. E. Ealick, Advances in multiple wavelength anomalous diffraction crystallography. Curr Opin Chem Biol 4, 495-499 (2000).
21. A. Vagin, A. Teplyakov, Molecular replacement with MOLREP. Acta Crystallogr D Biol Crystallogr 66, 22-25 (2010).
22. J. M. Guss et al., Phase determination by multiple-wavelength x-ray diffraction: crystal structure of a basic "blue" copper protein from cucumbers. Science 241, 806-811 (1988).
23. M. Benfatto et al., Multiple-scattering regime and higher-order correlations in x-ray-absorption spectra of liquid solutions. Phys Rev B Condens Matter 34, 5774-5781 (1986).
24. H. Walden, Selenium incorporation using recombinant techniques. Acta Crystallogr D 66, 352-357 (2010).
25. W. A. Hendrickson, C. M. Ogata, Phase determination from multiwavelength anomalous diffraction measurements. Method Enzymol 276, 494-523 (1997).
26. G. D. Fasman, Circular dichroism and the conformational analysis of biomolecules. The language of science (Plenum Press, New York, 1996), pp. ix, 738 p.
27. U. J. Meierhenrich et al., Circular dichroism of amino acids in the vacuum-ultraviolet region. Angew Chem Int Ed Engl 49, 7799-7802 (2010).
28. L. Whitmore, B. A. Wallace, Protein secondary structure analyses from circular dichroism spectroscopy: Methods and reference databases. Biopolymers 89, 392-400 (2008).
29. N. J. Greenfield, Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1, 2876-2890 (2006).
30. J. M. Bartlett, D. Stirling, A short history of the polymerase chain reaction. Methods Mol Biol 226, 3-6 (2003).
31. M. Uhlen, Affinity as a tool in life science. Biotechniques 44, 649-654 (2008).
32. S. Paul-Dauphin et al., Probing size exclusion mechanisms of complex hydrocarbon mixtures: The effect of altering eluent compositions. Energ Fuel 21, 3484-3489 (2007).
33. R. Tyagi, R. Lai, R. G. Duggleby, A new approach to ′megaprimer′ polymerase chain reaction mutagenesis without an intermediate gel purification step. Bmc Biotechnol 4, (2004).
34. L. H. Hansen, S. Knudsen, S. J. Sorensen, The effect of the lacY gene on the induction of IPTG inducible promoters, studied in Escherichia coli and Pseudomonas fluorescens. Curr Microbiol 36, 341-347 (1998).
35. A. Marbach, K. Bettenbrock, lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA. J Biotechnol 157, 82-88 (2012).
36. A. A. Watson, C. A. O′Callaghan, Crystallization and X-ray diffraction analysis of human CLEC-2. Acta Crystallogr Sect F Struct Biol Cryst Commun 61, 1094-1096 (2005).
37. M. D. Winn et al., Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67, 235-242 (2011).
指導教授 陳青諭(Chin-Yu Chen) 審核日期 2016-6-29
推文 facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu   
網路書籤 Google bookmarks   del.icio.us   hemidemi   myshare   

若有論文相關問題,請聯絡國立中央大學圖書館推廣服務組 TEL:(03)422-7151轉57407,或E-mail聯絡  - 隱私權政策聲明