請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48926完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 徐駿森(Chun-Hua Hsu) | |
| dc.contributor.author | Chia-Wei Chou | en |
| dc.contributor.author | 周家緯 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:11:40Z | - |
| dc.date.available | 2021-09-08 | |
| dc.date.copyright | 2016-09-08 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-22 | |
| dc.identifier.citation | 1. Abola, A.P, Abola, A. P., Willits, M. G., Wang, R. C. and Long, S. R. Reduction of adenosine-5'-phosphosulfate instead of 3'-phosphoadenosine-5'-phosphosulfate in cysteine biosynthesis by Rhizobium meliloti and other members of the family Rhizobiaceae. J Bacteriol, 1999. 181(17): p. 5280-7.
2. Berendt, U., Haverkamp, T., Prior, A. and Schwenn, J. D. Reaction mechanism of thioredoxin: 3'-phospho-adenylylsulfate reductase investigated by site-directed mutagenesis. Eur J Biochem, 1995. 233(1): p. 347-56. 3. Berndt, C, Lillig, C. H., Wollenberg, M., Bill, E., Mansilla, M. C., de Mendoza, D., Seidler, A. and Schwenn, J. D. Characterization and reconstitution of a 4Fe-4S adenylyl sulfate/phosphoadenylyl sulfate reductase from Bacillus subtilis. J Biol Chem, 2004. 279(9): p. 7850-5. 4. Bhave, D. P., Hong, J. A., Keller, R. L., Krebs, C., & Carroll, K. S. 'Iron-Sulfur Cluster Engineering Provides Insight into the Evolution of Substrate Specificity among Sulfonucleotide Reductases.' ACS Chem Biol, 2012. 7(2): 305-314. 5. Bick, J.A., Dennis, J. J., Zylstra, G. J., Nowack, J. and Leustek, T. Identification of a new class of 5'-adenylylsulfate (APS) reductases from sulfate-assimilating bacteria. J Bacteriol, 2000. 182(1): p. 135-42. 6. Carroll, K. S., Gao, H., Chen, H. Y., Stout, C. D., Leary, J. A., and Bertozzi, C. R. A conserved mechanism for sulfonucleotide reduction. PLoS Biol, 2005. 3(8): p. e250. 7. Carroll, K. S., Gao, H., Chen, H. Y., Leary, J. A., & Bertozzi, C. R. Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase: implications for substrate binding and catalysis. Biochemistry, 2005. 44(44): p. 14647-57. 8. Chartron, J., Shiau, C., Stout, C. D., & Carroll, K. S. 3'-Phosphoadenosine-5'-phosphosulfate reductase in complex with thioredoxin: a structural snapshot in the catalytic cycle. Biochemistry, 2007. 46(13): p. 3942-51. 9. Chartron, J., Carroll, K. S., Shiau, C., Gao, H., Leary, J. A., Bertozzi, C. R., and Stout, C. D. Substrate recognition, protein dynamics, and iron-sulfur cluster in Pseudomonas aeruginosa adenosine 5'-phosphosulfate reductase. J Mol Biol, 2006. 364(2): p. 152-69. 10. Chung, J. S., Noguera-Mazon, V., Lancelin, J. M., Kim, S. K., Hirasawa, M., Hologne, M., Knaff, D. B. Interaction domain on thioredoxin for Pseudomonas aeruginosa 5'-adenylylsulfate reductase. J Biol Chem, 2009. 284(45): p. 31181-9. 11. Corbett, E. L., Watt, C. J., Walker, N., Maher, D., Williams, B. G., Raviglione, M. C., and Dye, C. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med, 2003. 163(9): p. 1009-21. 12. Emsley, P. and K. Cowtan, Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr, 2004. 60(Pt 12 Pt 1): p. 2126-32. 13. Gao, H., et al., Noncovalent complexes of APS reductase from M. tuberculosis: delineating a mechanistic model using ESI-FTICR MS. J Am Soc Mass Spectrom, 2007. 18(2): p. 167-78. 14. 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. 15. Garman, E.F. and S. Doublie, Cryocooling of macromolecular crystals: optimization methods. Methods Enzymol, 2003. 368: p. 188-216. 16. Gonzalez.P and Baldeste.A. Involvement of Thioredoxin System in Reduction of Methionine Sulfoxide and Sulfate. J Biol Chem, 1970. 245(9): p. 2371-&. 17. Grimaldi, P., Ruocco, M. R., Lanzotti, M. A., Ruggiero, A., Ruggiero, I., Arcari, P., and Masullo, M. Characterisation of the components of the thioredoxin system in the archaeon Sulfolobus solfataricus. Extremophiles, 2008. 12(4): p. 553-62. 18. Hansch, R., Lang, C., Riebeseel, E., Lindigkeit, R., Gessler, A., Rennenberg, H., and Mendel, R. R. Plant sulfite oxidase as novel producer of H2O2: combination of enzyme catalysis with a subsequent non-enzymatic reaction step. J. Biol. Chem, 2006. 281: 6884–6888. 19. Holmgren, A., Thioredoxin and glutaredoxin systems. J Biol Chem, 1989. 264(24): p. 13963-6. 20. Khan, M.S., Haas, F.H., Allboje Samami, A., Moghaddas Gholami, A., Bauer, A., Fellenberg, K. Sulfite reductase defines a newly discovered bottleneck for assimilatory sulfate reduction and is essential for growth and development in Arabidopsis thaliana. Plant Cell, 2010. 22: 1216–1231. 21. Kim, S. K., Rahman, A., Bick, J. A., Conover, R. C., Johnson, M. K., Mason, J. T., and Knaff, D. B. 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. 22. Kim, S. K., Rahman, A., Mason, J. T., Hirasawa, M., Conover, R. C., Johnson, M. K., and Leustek, T. The interaction of 5'-adenylylsulfate reductase from Pseudomonas aeruginosa with its substrates. Biochim Biophys Acta, 2005. 1710(2-3): p. 103-12. 23. Kopriva, S., et al., Plant adenosine 5'-phosphosulfate reductase is a novel iron-sulfur protein. J Biol Chem, 2001. 276(46): p. 42881-6. 24. Kopriva, S., et al., The presence of an iron-sulfur cluster in adenosine 5'-phosphosulfate reductase separates organisms utilizing adenosine 5'-phosphosulfate and phosphoadenosine 5'-phosphosulfate for sulfate assimilation. J Biol Chem, 2002. 277(24): p. 21786-91. 25. Kopriva, S., et al., The putative moss 3'-phosphoadenosine-5'-phosphosulfate reductase is a novel form of adenosine-5'-phosphosulfate reductase without an iron-sulfur cluster. J Biol Chem, 2007. 282(31): p. 22930-8. 26. Lang, C., Popko, J., Wirtz, M., Hell, R., Herschbach, C., Kreuzwieser, J. et al. Sulphite oxidase as key enzyme for protecting plants against sulphur dioxide. Plant Cell Environ, 2007. 30: 447–455. 27. Leustek, T., Martin, M. N., Bick, J. A., and Davies, J. P. Pathways and Regulation of Sulfur Metabolism Revealed through Molecular and Genetic Studies. Annu Rev Plant Physiol Plant Mol Biol, 2000. 51: p. 141-165. 28. Martin, J.L., Thioredoxin--a fold for all reasons. Structure, 1995. 3(3): p. 245-50. 29. Newton, G.L. and R.C. Fahey, Mycothiol biochemistry. Arch Microbiol, 2002. 178(6): p. 388-94. 30. Osslund, T., C. Chandler, and I.H. Segel, ATP sulfurylase from higher plants : purification and preliminary kinetics studies on the cabbage leaf enzyme. Plant Physiol, 1982. 70(1): p. 39-45. 31. Rabeh, W.M. and P.F. Cook, Structure and mechanism of O-acetylserine sulfhydrylase. J Biol Chem, 2004. 279(26): p. 26803-6. 32. Renosto, F., P.A. Seubert, and I.H. Segel, Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Purification and kinetic characterization. J Biol Chem, 1984. 259(4): p. 2113-23. 33. Sassetti, C.M., D.H. Boyd, and E.J. Rubin, Comprehensive identification of conditionally essential genes in mycobacteria. Proc Natl Acad Sci U S A, 2001. 98(22): p. 12712-7. 34. 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. 35. 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. 36. Segel, I.H., F. Renosto, and P.A. Seubert, Sulfate-activating enzymes. Methods Enzymol, 1987. 143: p. 334-49. 37. Senaratne, R. H., De Silva, A. D., Williams, S. J., Mougous, J. D., Reader, J. R., Zhang, T. J., and Riley, L. W. 5'-Adenosinephosphosulphate reductase (CysH) protects Mycobacterium tuberculosis against free radicals during chronic infection phase in mice. Mol Microbiol, 2006. 59(6): p. 1744-53. 38. Seubert, P. A., Hoang, L., Renosto, F., and Segel, I. H. , ATP sulfurylase from Penicillium chrysogenum: measurements of the true specific activity of an enzyme subject to potent product inhibition and a reassessment of the kinetic mechanism. Arch Biochem Biophys, 1983. 225(2): p. 679-91. 39. 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. 40. Teschner, J., Lachmann, N., Schulze, J., Geisler, M., Selbach, K., Santamaria-Araujo, J. et al. (2010) A novel role for arabidopsis mitochondrial ABC transporter ATM3 in molybdenum cofactor biosynthesis. Plant Cell 22: 468–480. 41. Thauer, R.K., K. Jungermann, and K. Decker, Energy conservation in chemotrophic anaerobic bacteria. Bacteriol Rev, 1977. 41(1): p. 100-80. 42. Weber, M., Suter, M., Brunold, C., and Kopriva, S. 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. 43. Williams, S. J., Senaratne, R. H., Mougous, J. D., Riley, L. W., & Bertozzi, C. R. 5'-adenosinephosphosulfate lies at a metabolic branch point in mycobacteria. J Biol Chem, 2002. 277(36): p. 32606-15. 44. Yu, Z. H., Lemongello, D., Segel, I. H., and Fisher, A. J. Crystal structure of Saccharomyces cerevisiae 3'-phosphoadenosine-5'-phosphosulfate reductase complexed with adenosine 3',5'-bisphosphate. Biochemistry, 2008. 47(48): p. 12777-86. Zhang, Y., K. Post-Martens, and S. Denkin, New drug candidates and therapeutic targets for tuberculosis therapy. Drug Discov Today, 2006. 11(1-2): p. 21-7. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48926 | - |
| dc.description.abstract | 硫酸腺苷還原酶 (sulfonucleotide reductase ; SNR) ，為細菌、真菌及植物生合成生存必需之含硫化合物的系列反應中，將外界取得的 sulfate (SO42-) 進行硫同化 (sulfate assimilation)之關鍵反應步驟酵素。由於哺乳動物並不會進行此類的還原途徑，使得此酵素被視為新穎抗菌藥物開發的潛力目標，因此SNR的快速活性測試方法對於藥物的篩選有其必要性。在過去僅有兩種方法被使用於此酵素的活性測定，而且這兩種方法都必須使用到放射性元素標定，這些放射性方法最主要的缺點就是必須特別注意這些放射性元素的物質與廢棄物。本研究發展一新穎的非放射性酵素活性測定法，希望此簡單且價格低廉的比色法可有利於高通量的藥物篩選。
我們利用枯草桿菌的硫酸腺苷還原酶 (sulfonucleotide reductase from Bacillus subtilis, BsSNR) 進行研究與方法的開發，此一酵素有趣的是其兼具有 APS (adenosine 5’phosphosulfate) reductase 與 PAPS(3’phosphoadenosine 5’phosphosulfate) reductase 的活性，兩種基質皆能被催化。經由表現純化後所得到的重組蛋白呈現棕色來看，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 吸光光譜和鐵硫簇移除試驗，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) 的測定可以得知此種蛋白的 Tm 約在 56.1°C 左右。為獲得更詳盡的結構資訊，我們也利用已知結構作為模版對 BsSNR 進行結構模擬分析，藉由比較 BsSNR-APS 與 BsSNR-PAPS 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，我們提供了一個新的酵素活性測定方法，此一方法可以透過比色法的方式測定不論 PAPS 或者 APS 經由 SNR 反應後產生的亞硫酸根。我們也利用此方法測定 BsSNR 的酵素動力學參數，並且比較放射性元素標定方法之酵素動力學參數，這些結果顯示此方法於 SNR 相關的酵素研究與藥物篩選應用有一定的可靠性。 | zh_TW |
| dc.description.abstract | Sulfonucleotide reductase (SNR) catalyzes the first committed step in sulfate reduction for the biosynthesis of cysteine and is essential for survival in the latent phase of bacterial infection. However, metazoans do not possess the sulfate reduction pathway, which makes sulfonucleotide reductases being a promising target for drug development against human pathogens. As such, a rapid assay of SNR activity is valuable in drug screening. Only two methods are currently used to measure SNR activity, both involving radiolabeled material, however, the chief disadvantage of these assays is that special precautions are required due to the radioactive material and wastes involved. In this study, we developed a novel non-radioactive assay for monitoring SNR activity, and would like to apply this siple and cost-effective colorimetric method for high-throughput drug screening.
BsSNR (sulfonucleotide reductases from Bacillus subtilis) were used to develop the newly assay and study the substrates specificity of SNR, Interestingly, both PAPS (3'phosphoadenosine 5′-phosphosulfate) and APS (adenosine 5′-phosphosulfate) are substrates for BsSNR. Purified proteins in solution are shown brownish in color and proposed they should contain one [4Fe-4S] cluster per polypeptide chain. Data from ultraviolet-visible absorption spectroscopy would be collected to elucidate the nature of the prosthetic group containing properties. CD experiments showed that BsSNR possesses the Tm about 56.1 °C. To explain the substrates specificity, we got a homology modeling of BsSNR, and found the important residues involving in the binding of substrates. Taken together, we developed a novel SNR activity assay, which involves of either APS- or PAPS-dependent sulfite generation using colorimetric method. Based on the method, we determined the substrate selectivity and enzyme kinetic parameters of BsSNR. Our results reveal the current method is reliable with many potential applications in SNR studies and drug screening. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T11:11:40Z (GMT). No. of bitstreams: 1 ntu-105-R03623010-1.pdf: 8752579 bytes, checksum: da35c5d464752392a241a842bb95c2c8 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 摘要 I
Abstract II 縮寫表 VIII 壹、 前言 1 一、 Sulfonucleotide reductase (SNR) 硫酸腺苷還原酶的功能 1 二、 枯草桿菌(Bacillus subtilis)中的硫酸腺苷還原酶(SNR) 3 三、 潛力藥物開發目標 4 四、 二階段反應機制 (Two-step catalytic mechanism) 5 五、 酵素活性測定法 7 六、 研究目的 8 貳、實驗材料與方法 9 一、實驗材料 9 1. 蛋白質體表現與構築 9 2. 菌株及培養系統 9 3. Bacillus subtilis菌種 9 二、實驗方法 10 1. 蛋白表現載體的構築 10 2. 轉型作用 (transformation) 12 3. Colony PCR 12 4. DNA 膠體電泳分析 (agarose electrophoresis) 12 5. 質體DNA之抽取 13 6. DNA定序與比對 13 7. 蛋白質小量表現 13 8. 蛋白質大量表現 14 9. 蛋白質透析與濃縮 14 10. 膠體過濾層析法 (Gel Filtration Chromatography) 14 11. 蛋白質濃度測定 15 12. 圓二色光譜 (Circular Dichroism，CD) 實驗 16 13. UV-Vis吸收光譜測試 16 14. 蛋白質交聯測試 (crosslinking test) 16 15. 親緣關係 17 16. 酵素動力學方法 17 17. 胺基酸序列比對 18 18. 蛋白質結晶測試 18 19. 蛋白質結晶條件篩選 18 20. 蛋白質晶體形成條件之微調 19 21. 抗凍劑成分篩選與晶體繞射數據收集 19 參、實驗結果 21 一、BsSNR表現與純化 21 二、[4Fe-4S] 簇存在測定實驗 22 三、結構穩定性測試 22 四、蛋白質晶體培養 23 五、胺基酸序列比對 24 六、 BsSNR結構模型的建立 24 七、 BsSNR蛋白質結構與摺疊 25 八、 蛋白結構中的[4Fe-4S]結構 25 九、 基質 APS 與 PAP的辨認 26 肆、討論 28 一、Bacillus subtilis 中的 SNR 為一個具有 [4Fe-4S] cluster的PAPS reductase 28 二、新研發之酵素活性測定方法的重要性 29 三、BsSNR的酵素活性測定 30 四、BsSNR 晶體的培養 31 五、BsSNR 對於兩種受質的兼容性 32 伍、圖表 33 陸、參考文獻 63 柒、附錄 70 附錄一、溶液配方 70 附錄二、實驗中所用蛋白胺基酸組成及序列特性分析整理 75 附錄三、蛋白質載體資訊和構築示意圖 79 圖目錄 圖 1. 硫酸的還原代謝 (reductive sulfate assimilation) 途徑示意圖 33 圖 2. 硫酸腺苷還原酶比色法酵素活性試驗示意圖 34 圖 3. 亞硫酸根標準品之回歸曲線 35 圖 4. BsSNR的大量表現與 Ni2+親和性管柱純化結果 36 圖 5. BsTrx的大量表現與 Ni2+親和性管柱純化結果 37 圖 6. BsSNR Gel filtration 結果 38 圖 7. 純化到的 BsSNR 呈現棕色外觀 39 圖 8. UV-Vis吸收光譜 40 圖 9. BsSNR 在不同pH下的二級結構變化 41 圖 10. BsSNR 升溫時二級結構的變化 42 圖 11. Cross linking試驗 43 圖 12. BsSNR的最適反應溫度與受質選擇性 44 圖 13. BsSNR 對受質APS之酵素動力學 45 圖 14. BsSNR 對受質PAPS之酵素動力學 46 圖 15. BsTrx 第二階段反應之酵素動力學 47 圖 16. EcTrx 第二階段反應之酵素動力學 48 圖 17. ADP 影響 BsSNR 與 APS之結合 49 圖 18. ADP 影響 BsSNR 與 PAPS之結合 50 圖 19. BsSNR篩選出之可能晶體與繞射點圖 51 圖 20. BsSNR篩選出之可能晶體與繞射點圖 52 圖 21. BsSNR 模擬之立體結構 53 圖 22. 親緣關係圖 54 圖 23. SNR 胺基酸序列比對結果 55 圖 24. BsSNR 和 E.coli APS reductase 的活性區比較 56 圖 25. P-loop結構模擬圖 57 圖 26 . BsSNR 和 AMP 的結合模式分析 58 圖 27 . BsSNR 和 PAP 的結合模式分析 59 圖 28. 利用 ligplot 對 AMP 結合進行分析 60 圖 29. 利用 ligplot 對 PAP 結合進行分析 61 表目錄 表 1. 發展之比色法與放射性標定方法測量BsSNR酵素動力學參數的比較 62 | |
| dc.language.iso | zh-TW | |
| dc.subject | 活性測定 | zh_TW |
| dc.subject | 枯草桿菌 | zh_TW |
| dc.subject | 磷酸腺?還原? | zh_TW |
| dc.subject | activity assay | en |
| dc.subject | Bacillus subtilis | en |
| dc.subject | sulfonucleotide reductase | en |
| dc.title | 以結構分析與新發展之非放射性物質的酵素活性測定方法探討硫酸腺苷還原酶的受質選擇性 | zh_TW |
| dc.title | Understanding the substrate specificity of sulfonucleotide reductases based on structural analysis and newly developed non-radioactive assay | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林翰佳(Han-Jia Lin),詹迺立(Nei-Li Chan) | |
| dc.subject.keyword | 枯草桿菌,磷酸腺?還原?,活性測定, | zh_TW |
| dc.subject.keyword | Bacillus subtilis,sulfonucleotide reductase,activity assay, | en |
| dc.relation.page | 80 | |
| dc.identifier.doi | 10.6342/NTU201603351 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-22 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-105-1.pdf 未授權公開取用 | 8.55 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。