Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24062
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dc.contributor袁小琀zh_TW
dc.contributorHanna S. Yuanen_US
dc.contributor余靖zh_TW
dc.contributor張功耀zh_TW
dc.contributor李天雄zh_TW
dc.contributorChin Yuen_US
dc.contributorKung-Yao Changen_US
dc.contributorTien Hsiung Lien_US
dc.contributor.advisor周三和zh_TW
dc.contributor.advisorShan-Ho Chouen_US
dc.contributor.author廖淑如zh_TW
dc.contributor.authorLiao, Shu-Juen_US
dc.contributor.other中興大學zh_TW
dc.date2011zh_TW
dc.date.accessioned2014-06-06T07:21:54Z-
dc.date.available2014-06-06T07:21:54Z-
dc.identifierU0005-2107201016083800zh_TW
dc.identifier.citation1. Chae, H.Z., Robison, K., Poole, L. B., Church, G., Storz, G. & Rhee, S. G. (1994) Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes. Proc. Natl Acad. Sci. USA 91, 7017-7021. 2. Chae, H.Z., Chung, S. J. & Rhee, S. G. (1994) Thioredoxin-dependent peroxide reductase from yeast. J. Biol. Chem. 269, 27670-27678. 3. Costa Seaver, L. & Imlay, J.A. (2001) Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli. J. Bacteriol. 183,7173-7181. 4. Kawazu, S.-i., Komaki-Yasuda, K., Oku, H. & Kano, S. (2008) Peroxiredoxins in malaria parasites: parasiltologic aspects. Parasitol. Int. 57, 1-7. 5. Muller, S. (2004) Redox and antioxidant systems of the malaria parasite Plasmodium falciparum. Mol. Microbiol. 53, 1291-1305. 6. Hofmann, B., Hecht, H. J. & Flohe, L. (2002) Peroxiredoxins. Biol. Chem. 383, 347-364. 7. Fujii, J. & Ikeda, Y. (2002) Advances in our understanding of peroxiredoxin, a multifunctional, mammalian redox protein. Redox Rep. 7, 123-130. 8. Link, A.J., Robison, K. & Church, G. M. (1997) Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12. Electrophoresis 18, 1259-1313. 9. Moore, R.B., Mankad, M. V., Shriver, S. K., Mankad, V. N. & Plishker, G. A. (1991) Reconstitution of Ca2+-dependent K+ transport in erythrocyte membrane vesicles requires a cytoplasmic protein. J. Biol. Chem. 266, 18964-18968. 10. Chae, H.Z., Kim, H. J., Kang, S. W. & Rhee, S. G. (1999) Characterization of three isoforms of mammalian peroxiredoxin that reduce peroxides in the presence of thioredoxin. Diabetes Res. Clin. Pract. 45, 101-112. 11. Ellis, H.R. & Poole, L.B. (1997) Novel application of 7-chloro-4-nitrobenzo-2-oxa -1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase. Biochemistry 36, 15013-15018. 12. Choi, H.-J., Kang, S. W., Yang, C. H., Rhee, S. G. & Ryu, S. E. (1998) Crystal structure of a novel human peroxidase enzyme at 2.0A ° resolution. Nat. Struct. Biol. 5, 400-406. 13. Wood, Z.A. Poole, L. B., Hantgan, R. R. & Karplus, P. A. (2002) Dimers to doughnuts: redox-sensitive oligomerization of 2-cysteine peroxiredoxins. Biochemistry 41, 5493-5504. 14. Seo, M.S., Kang, S. W., Kimi, K. I., Baines, C., Lee, T. H. & Rhee, S. G. (2000) Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate. J. Biol. Chem. 275, 20346-20354. 15. Wood, Z. A., Schroder, E., Harris, J. R. & Poole, L. B. (2003) Structure, mechanism and regulation of peroxiredoxins. Trends Biochem. Sci. 28, 32-40. 16. Neidhardt, F.C., Vaughn, V., Phillips, T. A. & Bloch, P. L. (1983) Gene-protein index of Escherichia coli K-12. Microbiol. Rev. 47, 231-284. 17. Jeong,W., Cha, M. -K. & Kim, I.-H. (2000) Thioredoxin-dependent hydroperoxide peroxidase activity of bacterioferritin comigratory protein (BCP) as a new member of the thio-specific antioxidant protein (TSA)/alkyl hydroperoxide peroxidase C (AhpC) family. J. Biol. Chem. 275, 2924-2930. 18. Wang, G., Olczak, A. A. Walton, J. P. & Maier, R. J. (2005) Contribution of the Helicobacter pylori thiol peroxidase bacterioferritin comigratory protein to oxidative stress resistance and host colonization. Infect. Immun. 73, 378-384. 19. Kong, W., Shiota, S., Shi, Y., Nakayama, H. & Nakayama, K. (2000) A novel peroxiredoxin of the plant Sedum lineare is a homologue of Escherichia coli bacterioferritin co-migratory protein (Bcp). Biochem. J. 351, 107-114. 20. Sarma, G. N., Nickel, C., Rahlfs, S., Fischer, M., Becker,K. & Karpuls, P. A. (2005) Crystal structure of a novel Plasmodium falciparum 1-Cys peroxiredoxin. J. Mol. Biol. 346, 1021-1034. 21. Li, S., Peterson, N. A., Kim, M. Y., Kim, C. Y., Hung, L. W., Yu, M., Lekin, T., Segelke, B. W., Lott1, J. S. & Baker, E. N. (2005) Crystal structure of AhpE from Mycobacterium tuberculosis, a 1-Cys peroxiredoxin. J. Mol. Biol. 346, 1035-1046. 22. Schroder, E., Littlechild, J. A., Lebedev, A. A.,Errington, N., Vagin, A. A. & Isupov, M. N. (2000) Crystal structure of decameric 2-Cys peroxiredxin from human erythrocytes at 1.7Å resolution. Structure 8, 605-615. 23. Mizohata, E., Sakai, H., Fusatomi, E., Terada, T.,Murayama, K., Shirouzu, M. & Yokoyama, S. (2005) Crystal structure of an Archaeal peroxiredoxin from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1. J. Mol. Biol. 354, 317-329. 24. Matsumura, T., Okamato, K., Iwahara, S. -i., Hori, H.,Takahashi, Y., Nishino, T. & Abe, Y. (2008) Dimer-oligomer interconversion of wild-type and mutant rat 2-Cys peroxiredoxin: Disulfide formation at dimer-dimer interfaces is not essential for decamerization. J. Biol. Chem. 283, 284-293. 25. Guimarzes, B. C., Souchon, H., Honore, N., Saint-Joanis, B., Brosch, R., Shepard, W., Cole, S. T. & Alzari, P. M. (2005) Structure and mechanism of the alkyl hydroperoxidase AhpC, a key element of the Mycobacterium tuberculosis defense system against oxidative stress. J. Biol. Chem. 280, 25735-25742. 26. Limauro, D., Pedone, E., Gaildi, I. & Bartolucci, S. (2008) Peroxiredoxins as cellular guardians in Sulfolobus solfataricus- characterizatin of Bcp1, Bcp3, and Bcp4. FEBS J. 275, 2067-2077. 27. Smeets, A., Marchand, C., Linard, D., Knoops, B. & Declercq, J. -P. (2008) The crystal structures of oxidized forms of human peroxiredloxin 5 with an intramolecular disulfide bond confirm the proposed enzymatic mechanism for atypical 2-Cys peroxiredoxins. Arch. Biochem. Biophys. 477, 98-104. 28. Evrard, C., Capron, A., Marchand, C., Clippe, A.,Wattiez, R., Soumillion, P., Knoops, B. & Declercq, J. -P. (2004) Crystal structure of a dimeric oxidized form of human peroxiredoxin 5. J. Mol. Biol. 337, 1079-1090. 29. Choi, J., Choi, S., Chon, J. K., Choi, J., Cha, M.-K., Kim,I. -H. & Shin,W. (2005) Crystal structure of the C107S/C112S mutant of yeast nuclear 2-Cys peroxiredoxin. Proteins: Struct. Funct. Bioinformatics 61, 1146-1149. 30. Choi, J., Choi, S., Choi, J., Cha, M.-K., Kim, I. -H. & Shin, W. (2003) Crystal structuer of Escherichia coli thiol peroxidase in the oxidized state: Insight into intramolecular disulfide formation and substrate binding in atypical 2-Cys peroxiredoxins. J. Biol. Chem. 278, 49478-49486. 31. Aslanidis C. and de Jong, P.C. ( 1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069-6074. 32. Wu, Y.-Y., Chin, K. -H., Chou, C. -C., Lee, C. -C., Shr H. -L., Gao F. P., Lyu, P. -C., Wang, Andrew H.-J. & Chou, S. -H. (2005) Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of XC847, a 30-50 oligoribonuclease from Xanthomonas campestris. Acta. Cryst. F. 16, 902-905. 33. Vandeyar, M. A., Weiner, M. P., Hutton, C. J. & Batt, C. A. (1988) A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene 65, 129-133. 34. Otwinowski, Z. & Minor, W. (1997) Processing of the X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-326. 35. Terwilliger, T. C. & Berendzen, J. (1999) Automated MAD and MIR structure solution. Acta Crystallogr. D. 55, 849-861. 36. McRee, D. E. (1999) XtalView/Xfit - a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156-165. 37. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. & Warren, G. L. (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D. 54, 905-921. 38. Vagin, A. & Teplyakov, A. (1997) MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022-1025. 39. Wu, G., Robertson, D. H., Brooks, C. L. & Vieth, M. (2003) Detailed analysis of grid-based molecular docking: a case study of CDOCKER-a CHARMm-based MD docking algorithm. J Comp Chem. 24, 1549-1562. 40. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680. 41. Barton, G. J. (1993) ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng. 6, 37-40.zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/24062-
dc.description.abstract需氧生物藉由氧氣以進行體內各項機能的運作,但代謝過程中體內的物質會發生氧化反應而產生過氧化物(peroxides),造成細胞的死亡。因此生物體具有多種清除過氧化物的防禦性酵素,例如:超氧化物歧化酶(superoxide dismutase,簡稱SOD)、過氧化氫酶(Catalase,CAT)、麩胱甘肽過氧化酶(Glutathione peroxidase, GPx)及過氧化氧化還原酶(peroxiredoxins 簡稱 Prxs)。新發現的Prxs氧化還原酶,為硫醇特異性抗氧化蛋白,又稱為thioredoxin peroxidases 及alkyl-hydroperoxide-reductase -C22 proteins。Prxs 依照其所參與氧化還原反應的半胱胺酸(Cysteine)之數目與位置可分為1-Cys Prxs、典型的2-Cys Prxs 及非典型的2-Cys Prxs。Prx 蛋白在不同的生物體中有不同數目的異構型,在哺乳動物細胞中,目前發現有六種(PrxI-VI),而在大腸桿菌中則有三種(AhpC、p20、BCP)。許多典型的2-Cys Prxs 及1-Cys Prxs 的三級結構己經被解析出來,進而瞭解其作用機制。但在非典型的2-Cys Prxs 包含大腸桿菌中之BCP (Bacterioferritin comigratory protein)蛋白的三級結構及作用機制仍不太清楚。在此我們同時解析了XcBCP 氧化態(Intra_SS) ; XcBCP 還原態(Free_SH)及XcBCP 含ligand(Inter-SH)的蛋白晶體結構。分析此三結構的結果指出,其三級結構除了具有高度保留的活化中心結構 ( thioredoxin fold),並進一步發現,其為一新型的非典型2-Cys Prxs 蛋白。並藉由與ligand (DNS)的共結晶,我們可以進一步推測XcBCP蛋白與alkyl peroxide substrates 的作用機制。XcBCP 蛋白藉由N 端的氧化半胱胺酸(peroxidatic cysteine)與C 端的還原半胱胺酸(resolving cysteine),將過氧化物還原。而本身成為具有分子內雙硫鍵的氧化態蛋白,並藉由硫氧化還原蛋(Trx)及其還原酵素(TrxR)將氧化態BCP 蛋白還原成還原態,進而繼續還原下一個過氧化物。zh_TW
dc.description.abstractAerobically growing cells are constantly challenged by reactive oxygen species (ROS), potent oxidants capable of damaging cellular components. To protect against the toxicity of ROS, aerobic organisms are equipped with an array of defensive enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and Peroxiredoxin (Prx). Peroxiredoxins (Prxs), also termed the thioredoxin peroxidases and alkyl-hydroperoxide-reductase-C22 protein have received considerable attention in recent years as a new and expanding family of thiol-speific antioxidant proteins. Prxs are classified into three major subfamilies, the 1-Cys, the typical 2-Cys, and the atypical 2-Cys Prx, based on the number and position of Cys residue that participate in catalysis. Many organisms produce more than one isoform of Prx, for examples, mammalian cells express six isoforms of Prx (Prx I to VI) and E coli has three isoforms (AhpC, p20, and BCP). The tertiary structures of several typical 2-Cys and 1-Cys Prx have been determined. However, atypical 2-Cys Prx, including the bacterioferritin comigratory protein (BCP), are less well characterized. Here, we report the crystal structures of XcBCP, a novel member of the atypical 2-Cys Prx from the plant pathogen Xanthomonas campestris, in three different states, the Free_SH state, Intra_SSstate, and Inter _SS state. The oxidoreduction pathway of XcBCP has been obtained from trapped intermediate/ligand complex structures. The XcBCP contains the conserved CP-SH at the N terminus that is oxidized to sulfenic acid (CP-SOH) during the catalytic reaction. The CP-SOH is reduced back to CP-SH by a resolving CR-SH located at the C terminus end of the same subunit to form an intramolecular disulfide bond. The intramolecular disulfide bond of XcBCP can be shuttled back to thiol groups by thioredoxin (XcTrx) to complete the catalytic cycle.en_US
dc.description.tableofcontents中文摘要 ..................................................................................................................................... I Abstract....................................................................................................................................... II 目錄............................................................................................................................................ III 圖表目錄....................................................................................................................................V 第一章 前言............................................................................................................................... 1 第二章 材料與方法.................................................................................................................. 7 一、構築表現重組蛋白之載體.................................................................................. 7 二、定點突變(Site-Directed Mutagenes i s ) ............................................... 9 三、菌種之保存........................................................................................................ 10 四、蛋白質的大量表現以及純化............................................................................ 11 (一)誘發大量蛋白質表現................................................................................. 11 (二)以親和性管柱(Nickel column)進行蛋白質初步純化................... 12 (三)TEV 蛋白水解酶(Tobacco Etch Virus protease)作用............ 12 (四)陰離子交換樹脂純化................................................................................. 13 (五)分子篩管柱純化......................................................................................... 13 (六)蛋白質濃度測定......................................................................................... 14 五、蛋白質的活性分析............................................................................................ 14 六、XcBCP 蛋白質結晶實驗.................................................................................... 15 (一)結晶條件的初步篩選................................................................................. 15 (二)微調結晶條件............................................................................................. 16 IV 七、X-ray 晶體繞射實驗.......................................................................................... 16 八、蛋白質與小分子嵌合的研究分析(protein-ligand docking)............. 18 第三章 結果............................................................................................................................. 19 一、XC2714(XcBCP)蛋白的表現及純化........................................................... 19 (一)XcBCP 蛋白的親和性管柱純化................................................................ 19 (二)XcBCP 蛋白的陰離子交換樹脂純化........................................................ 20 (三)XcBCP 蛋白的分子篩管柱純化................................................................ 20 二、XcBCP 蛋白的活性分析.................................................................................... 20 三、結晶條件的篩選................................................................................................ 22 四、X-ray 繞射數據的分析...................................................................................... 24 五、XcBCP 蛋白之整體結構.................................................................................... 25 (一)無雙硫鍵階段(Free_SH State) ....................................................... 25 (二)分子間雙硫鍵階段(Inter_SS State)............................................... 26 (三)分子內雙硫鍵階段(Intra_SS State)............................................... 27 六、XcBCP 蛋白之活化中心與受質結合區............................................................ 28 第四章 討論............................................................................................................................. 30 一、XC2714 為非典型2-Cys 過氧化氧化還原酶家族的BCP 蛋白..................... 30 二、比較XcBCP、ApBCP 及EcTPX 的蛋白質結構.................................. 31 三、XcBCP 蛋白進行氧化還原時的結構變化........................................................ 32 四、XcBCP 蛋白的氧化還原作用機制.................................................................... 34 第五章 參考文獻.................................................................................................................... 36zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物化學研究所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2107201016083800en_US
dc.subjectperoxide oxidoreduction pathwayen_US
dc.subject過氧化氧化還原酶十字花科黑腐病zh_TW
dc.subjectBCPen_US
dc.subjectPrxsen_US
dc.subjectatypical 2-Cysen_US
dc.subjectBCP蛋白zh_TW
dc.subject非典型2-Cys過氧化氧化還原酶zh_TW
dc.title探討Xanthomonas campestris pv. campestris BCP (bacterioferritin comigratory protein)蛋白質結構及 還原烷基過氧化物的機制zh_TW
dc.titleInsights into the alkyl peroxide reduction pathway of Xanthomonas campestris bacterioferritin comigratory protein from the trapped intermediate-ligand complex structuresen_US
dc.typeThesis and Dissertationzh_TW
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