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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 徐駿森(Chun-Hua,Hsu) | |
| dc.contributor.author | I-Shao Chen | en |
| dc.contributor.author | 陳奕劭 | zh_TW |
| dc.date.accessioned | 2021-06-15T00:15:36Z | - |
| dc.date.available | 2016-08-19 | |
| dc.date.copyright | 2011-08-19 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-15 | |
| dc.identifier.citation | 1. Society for General Microbiology. Symposium (42nd : 1988 : University of Southampton), J.A. Cole, and S.J. Ferguson, The nitrogen and sulphur cycles. 1988, Cambridge ; New York: Cambridge University Press. xii, 490 p.
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Bick, J.A., et al., Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria. J Bacteriol, 2000. 182(1): p. 135-42. 8. Berendt, U., et al., Reaction mechanism of thioredoxin: 3'-phospho-adenylylsulfate reductase investigated by site-directed mutagenesis. Eur J Biochem, 1995. 233(1): p. 347-56. 9. Schwenn, J.D., F.A. Krone, and K. Husmann, Yeast PAPS reductase: properties and requirements of the purified enzyme. Arch Microbiol, 1988. 150(4): p. 313-9. 10. Rabeh, W.M. and P.F. Cook, Structure and mechanism of O-acetylserine sulfhydrylase. J Biol Chem, 2004. 279(26): p. 26803-6. 11. Leustek, T., et al., Pathways and Regulation of Sulfur Metabolism Revealed through Molecular and Genetic Studies. Annu Rev Plant Physiol Plant Mol Biol, 2000. 51: p. 141-165. 12. Corbett, E.L., et al., The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med, 2003. 163(9): p. 1009-21. 13. Zhang, Y., K. 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Weber, M., et al., Sulfate assimilation in higher plants characterization of a stable intermediate in the adenosine 5'-phosphosulfate reductase reaction. Eur J Biochem, 2000. 267(12): p. 3647-53. 25. Gonzalez.P and Baldeste.A, Involvement of Thioredoxin System in Reduction of Methionine Sulfoxide and Sulfate. Journal of Biological Chemistry, 1970. 245(9): p. 2371-&. 26. Holmgren, A., Thioredoxin and glutaredoxin systems. J Biol Chem, 1989. 264(24): p. 13963-6. 27. Martin, J.L., Thioredoxin--a fold for all reasons. Structure, 1995. 3(3): p. 245-50. 28. Chartron, J., et al., 3'-Phosphoadenosine-5'-phosphosulfate reductase in complex with thioredoxin: a structural snapshot in the catalytic cycle. Biochemistry, 2007. 46(13): p. 3942-51. 29. Carroll, K.S., et al., Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase: implications for substrate binding and catalysis. Biochemistry, 2005. 44(44): p. 14647-57. 30. Chung, J.S., et al., Interaction domain on thioredoxin for Pseudomonas aeruginosa 5'-adenylylsulfate reductase. J Biol Chem, 2009. 284(45): p. 31181-9. 31. She, Q., et al., The complete genome of the crenarchaeon Sulfolobus solfataricus P2. Proc Natl Acad Sci U S A, 2001. 98(14): p. 7835-40. 32. Zillig, W., et al., The Sulfolobus-Caldariella Group - Taxonomy on the Basis of the Structure of DNA-Dependent Rna-Polymerases. Archives of Microbiology, 1980. 125(3): p. 259-269. 33. Garman, E.F. and S. Doublie, Cryocooling of macromolecular crystals: optimization methods. Methods Enzymol, 2003. 368: p. 188-216. 34. Garman, E.F. and R.L. Owen, Cryocooling and radiation damage in macromolecular crystallography. Acta Crystallogr D Biol Crystallogr, 2006. 62(Pt 1): p. 32-47. 35. McCoy, A.J., et al., Phaser crystallographic software. J Appl Crystallogr, 2007. 40(Pt 4): p. 658-674. 36. Emsley, P. and K. Cowtan, Coot: model-building tools for molecular graphics. 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Biochim Biophys Acta, 2005. 1710(2-3): p. 103-12. 42. Kim, S.K., et al., Properties of the cysteine residues and iron-sulfur cluster of the assimilatory 5'-adenylyl sulfate reductase from Pseudomonas aeruginosa. Biochemistry, 2004. 43(42): p. 13478-86. 43. Savage, H., et al., Crystal structure of phosphoadenylyl sulphate (PAPS) reductase: a new family of adenine nucleotide alpha hydrolases. Structure, 1997. 5(7): p. 895-906. 44. Rittle, J. and M.T. Green, Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics. Science, 2010. 330(6006): p. 933-7. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41309 | - |
| dc.description.abstract | 硫酸磷酸腺苷還原酶 (sulfonucleotide reductase) ，為一種將外界取得的 sulfate (SO42-) 進行還原代謝 (reductive sulfate assimilation) 之酵素。細菌、真菌及植物進行生合成 cysteine 的系列反應中，此酵素為第一個關鍵反應步驟，然而其反應機制仍然有許多未知的部份尚待釐清。此外，哺乳動物並不會進行此類的還原途徑，這也使得此酵素被視為新穎抗病菌藥物開發的潛力目標。
為了進一步闡明此類酵素的催化機制，本研究針對硫葉菌的硫酸磷酸腺苷還原酶 (sulfonucleotide reductase from Sulfolobus solfataricus, ssSNRase) 進行研究分析。經由表現純化到的重組蛋白呈現棕色，暗示了蛋白中可能含有金屬錯化物基團在其中，再藉由 UV-vis 吸光光譜和 X 射線螢光掃描分析，我們可以推測 [4Fe-4S] 簇輔基團的存在。此外，利用圓二色光譜儀 (circular dichroism) 測定可以得知此種蛋白的 Tm 在 82°C 以上，而將 [4Fe-4S] 簇移除則會降低此酵素的結構穩定性。為獲得更詳盡的結構資訊，我們進行 ssSNRase 與其產物 (adenosine 5’-monophosphate, AMP) 和基質 (adenosine 5’-phosphosulfate, APS) 複合物的共結晶實驗，並且成功解出其晶體結構，解析度分別為 1.97 及 2.03 Å。在這兩種不同催化階段的結構中，可以看到含有活性必要殘基 (residue) cysteine 231 的羧基端尾巴片段，被一個架構完整的氫鍵網絡固定在其酵素活性區上方的溝槽之中。藉由比較 ssSNRase-APS 與 ssSNRase-AMP 兩種不同複合物結構，我們發現到數個參與催化活性過程中的重要殘基並且推測出其功能。根據本篇實驗的結果，我們提供更多藉由酵素結構變化所調控的 SNRase 活性的相關訊息，也進一步的對其羧基端尾巴開闔的動態變化與調控機制提出更完整的註解。 | zh_TW |
| dc.description.abstract | The first committed step of reductive sulfate assimilation in the biosynthesis of cysteine in pathogenic bacteria is catalyzed by sulfonucleotide reductases (SNRases). However, mammals do not possess the sulfate reduction pathway, which makes sulfonucleotide reductases being a promising target for drug development against human pathogens. To elucidate the catalytic mechanism, ssSNRase (sulfonucleotide reductase from Sulfolobus solfataricus) was studied by a combination of biochemical, spectroscopic, and crystallographic approaches. Purified ssSNRase protein in solution is shown brownish in color and proposed it should contain one [4Fe-4S] cluster per polypeptide chain. Data from ultraviolet-visible absorption spectroscopy and X-ray fluorescence scan were collected to elucidate the nature of the prosthetic group containing property. CD experiments showed that ssSNRase possesses high melting temperature around 82°C, which consistent with the physiological growth environment of Sulfolobus solfataricus. Crystal structure of ssSNRases in complex with its product AMP and its substrate APS were solved to 1.97 and 2.03 Å, respectively. Residues that are essential for catalytic activity could be elucidated by comparing substrate-product substituted active sites and the possible function of these residues were also proposed. C-terminus tail containing active Cys231 residue in an organized H-bonding network with the channel of enzyme were both observed. By these two snapshots of ssSNRase catalytic states, we can provide more information about the C-terminus tail displacement and the mobility model of the C-terminal tail during APS reduction can be modified. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T00:15:36Z (GMT). No. of bitstreams: 1 ntu-100-R98623010-1.pdf: 12559726 bytes, checksum: 057d2531189a73edc4612d125f662647 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 目 錄
謝誌 i Abstract iii 目 錄 iv 圖目錄 vii 表目錄 ix 縮寫表 x 壹、前言 1 一、Sulfonucleotide reductase (SNRase) 的功能 1 二、藥物開發潛力目標 1 三、兩大種類的 sulfonucleotide reductase 2 四、二階段反應機制 (Two step mechanism) 3 五、Sulfolobus solfataricus 硫葉菌 3 六、研究目的 4 貳、實驗材料與方法 5 一、實驗材料 5 1. 蛋白質體表現與構築 5 2. 菌株及培養系統 5 3. Sulfolobus soltartaricus菌種 5 4. 蛋白晶體形成篩選套組 5 二、實驗方法 6 1. 轉形作用 (transformation) 6 2. 質體DNA之抽取 6 3. 蛋白表現載體的構築 6 4. DNA 膠體電泳分析 (agarose electrophoresis) 8 5. DNA 定序與比對 8 6. SDS 蛋白質膠體電泳分析 (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) 8 7. 蛋白質小量表現 9 8. 蛋白大量表現 9 9. 蛋白質純化 9 10. 蛋白質透析與濃縮 10 11. 蛋白質濃度測定 10 12. 圓二色光譜 (Circular Dichroism，CD) 實驗 11 13. UV-vis吸收光譜測試 11 14. 胺基酸序列比對 11 15. 羧基端活動性測試 11 16. 酵素活性測試 12 三、X-ray 晶體繞射技術解析蛋白質結構 12 四、X-ray 晶體繞射實驗方法 13 1. 蛋白質結晶測試 13 2. 蛋白質結晶條件篩選 13 3. 蛋白質晶體形成條件之微調 13 4. 篩選適合之抗凍劑 13 5. X-ray 晶體繞射數據收集及處理 14 6. 晶格中所包含的分子數分析 14 7. 蛋白結構建立 15 8. 蛋白結構模型修正 15 9. 結構確校 16 參、實驗結果 18 一、ssSNRase 表現與純化 18 二、[4Fe-4S] 簇存在測定實驗 19 三、結構穩定性測試 19 四、蛋白質晶體培養 20 五、Fe 的 K-edge 螢光掃描實驗 21 六、X-ray繞射數據分析與單位晶格判斷 21 七、晶格中所包含的分子數分析 22 八、蛋白結晶結構的建立 22 九、ssSNRase 蛋白結構摺疊 22 十、胺基酸序列比對 23 十一、蛋白結構當中的 [4Fe-4S] 簇 23 十二、反應物APS的辨認 24 十三、羧基端尾巴的存在狀態分析 25 十四、羧基端活動性測試 25 十五、ssTrxA1、ssTrxA2、ssTrxB3 蛋白表現載體之構築 26 十六、ssTrxA1、ssTrxA2、ssTrxB3 蛋白表現、純化結果 26 十七、酵素活性測試 27 肆、討論 28 一. S.solfataricus 的 sulfonucleotide reductase 是屬於 APS reductase 28 二. [4Fe-4S] 簇在 ssSNRase 中扮演的角色 28 三. ssSNRase 複合體結構特殊性之探討 29 四. 結構所處狀態 30 五. 調控羧基端尾巴片段移動力的機制 31 六. 修正的酵素反應機制 32 伍、圖表 33 陸、參考文獻 72 柒、附錄 75 | |
| dc.language.iso | zh-TW | |
| dc.subject | 蛋白質結晶學 | zh_TW |
| dc.subject | 還原酶 | zh_TW |
| dc.subject | 硫葉菌 | zh_TW |
| dc.subject | 催化機制 | zh_TW |
| dc.subject | 複合體結構 | zh_TW |
| dc.subject | 硫酸磷酸腺苷 | zh_TW |
| dc.subject | sulfonucleotide reductases | en |
| dc.subject | catalytic mechanism | en |
| dc.subject | protein crystallography | en |
| dc.subject | Sulfolobus solfataricus | en |
| dc.subject | crystal structure | en |
| dc.title | 硫葉菌硫酸磷酸腺苷還原酶與其基質及產物複合體提供催化機制的結構基礎層面探討 | zh_TW |
| dc.title | Sulfonucleotide reductase in complexes with substrate and product reveals the structural snapshots in the catalytic cycle | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 詹迺立,洪慧芝 | |
| dc.subject.keyword | 硫葉菌,硫酸磷酸腺苷,還原酶,催化機制,蛋白質結晶學,複合體結構, | zh_TW |
| dc.subject.keyword | Sulfolobus solfataricus,sulfonucleotide reductases,catalytic mechanism,protein crystallography,crystal structure, | en |
| dc.relation.page | 84 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-08-15 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 農業化學研究所 | zh_TW |
| 顯示於系所單位： | 農業化學系 | |
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