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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44852完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王惠鈞(Andrew H.-J. Wang) | |
| dc.contributor.author | Wei-Lin Huang | en |
| dc.contributor.author | 黃威霖 | zh_TW |
| dc.date.accessioned | 2021-06-15T03:56:29Z | - |
| dc.date.available | 2010-06-30 | |
| dc.date.copyright | 2010-06-30 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-06-21 | |
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Russell, The glutamine cyclotransferase reaction of Streptococcus bovis: a novel mechanism of deriving energy from non-oxidative and non-reductive deamination. FEMS Microbiol Lett, 1993. 111(2-3): p. 263-8. 69. McCracken, A.W. and C.U. Mauney, Identification of Corynebacterium diphtheriae by immunofluorescence during a diphtheria epidemic. J Clin Pathol, 1971. 24(7): p. 641-4. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44852 | - |
| dc.description.abstract | 麩酸胺環化酵素(EC 2.3.2.5)負責催化許多蛋白質及胜肽的氨端焦麩氨酸的形成，這個反應亦為許多生物活性分子成熟的重要步驟。在許多動植物中，具有麩酸胺環化酵素功能的蛋白質都已被確認，惟獨在細菌之中尚未發現；因此本篇論文中，我們研究從植物致病菌—十字花科蔬菜黑腐病菌( Xc )之中得到的第一種細菌麩酸胺環化酵素。這個酵素的晶體結構也已解出並將解析度改進到 1.44-A 。此酵素結構顯示其為一個五葉螺旋槳型式，並與已發表的木瓜麩酸胺環化酵素擁有類似架構，但與其相比仍有些序列缺失以及構型上的改變。
相對於木瓜麩酸胺環化酵素的結構，Xc 麩酸胺環化酵素的活性區位擁有較寬的疏水性口袋，但此活性區位的可及性卻被一個角色可能類似於口蓋的突出環形結構所調節。酵素活性分析顯示， Xc 麩酸胺環化酵素僅有木瓜麩酸胺環化酵素 3 % 活性；重疊兩個結構的比較結果發現，在木瓜麩酸胺環化酵素上的一個活性區域胺基酸－骨胺酰酸，在 Xc 麩酸胺環化酵素中被置換成了第 45 號骨胺酸，然而，細菌麩酸胺環化酵素的序列之中這個位置大多都是骨胺酰酸。這個點突變讓 Xc 麩酸胺環化酵素的活性增進了將近十倍之多，但將此胺基酸點突變成丙氨酸卻造成酵素活性的下降。這個改變也顯示了此胺基酸在催化機制上扮演重要的角色。更進一步的點突變研究也如同前人研究所假設的一樣，支持了第 89 號骨胺酸的催化角色，也更進一步的確認在受質結合位附近的一些保守胺基酸的重要性。 相對於木瓜麩酸胺環化酵素極高的穩定性，儘管 Xc 麩酸胺環化酵素與其擁有相類似的架構，仍然表現了對於鹽酸胍、極端酸鹼值以及熱失活的弱抵抗力。基於對此兩結構的比較，Xc 麩酸胺環化酵素的低穩定性可能緣自於β-螺旋槳型結構中缺少一個雙硫鍵鍵結，以及一些氫鍵的差異。這些結果增進了我們對第一型麩酸胺環化酵素的催化機制以及其異乎尋常之穩定性的了解，對此類酵素未來的應用助益良多。 | zh_TW |
| dc.description.abstract | Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the formation of pyroglutamate (pGlu) at the N-terminus of many proteins and peptides, a critical step for the maturation of these bioactive molecules. Proteins having QC activity have been identified in animals and plants, but not in bacteria. In this research, we report the first bacterial QC from the plant pathogen Xanthomonas campestris (Xc). The crystal structure of the enzyme was solved and refined to 1.44-A resolution. The structure shows a five-bladed beta-propeller and exhibits a similar scaffold to the published structure of papaya QC (pQC), but with some sequence deletions and conformational changes.
In contrast to the pQC structure, the active site of XcQC has a wider substrate binding pocket, but its accessibility is modulated by a protruding loop acting as a flap. Enzyme activity analyses showed that the wild type XcQC possesses only 3% QC activity of pQC. Superposition of those two structures revealed that an active-site glutamine residue in pQC is substituted by a glutamate (Glu45) in XcQC, although the position 45 is mostly a glutamine in bacterial QC sequences. The E45Q mutation increased the QC activity by an order of magnitude, but the mutation E45A led to a drop in the enzyme activity, indicating the critical catalytic role of this residue. Further mutagenesis studies support the catalytic role of Glu89 as proposed previously and confirm the importance of several conserved amino acids around the substrate-binding pocket. XcQC was shown to be weakly resistant to guanidine hydrochloride, extreme pH, and heat denaturations, in contrast to the extremely high stability of pQC, despite their similar scaffold. On the basis of structure comparison, the low stability of XcQC may be attributed to the absence of a disulfide linkage and some hydrogen bonds in the closure of beta-propeller structure. These results significantly improve our understanding of the catalytic mechanism and extreme stability of type I QCs, which will be useful in the further applications of QC enzymes. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T03:56:29Z (GMT). No. of bitstreams: 1 ntu-99-R97b46006-1.pdf: 10493948 bytes, checksum: 9b52a930fae42c2ecf6e857b5d8f9df5 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | List of Figures………………………………………………………………...…….. i
List of Tables…………………………………………….………….…………...….iii 中文摘要……………………………………………………………………….…....iv Abstract…………...…………………………………..……….……………….…... v Introduction………………………………………………………………………….1 Material and Methods…….………………………………………………………... 7 2.1 Protein expression and purification…...............................................................8 2.11 Xanthomonas campestris glutaminyl cyclase (XcQC)…….……......…...8 2.12 The mutants of XcQC……....……………………….…..…………...…..9 2.13 Cloning of an engineered XcQC………………………………………...9 2.2 Crystallization and X-ray data collection..........................................................9 2.3 Structure determination and refinement……………………………………..10 2.4 Molecular modeling and substrate binding simulation.……………………...11 2.5 Enzyme activity assay…………………………….…....…...............…….….11 2.6 Enzyme kinetic assay .….……….……….……….……….…………...….…12 2.7 Enzyme characters evaluation at different conditions………….……………13 2.71 Enzyme activities under various pH and temperature……………….....13 2.72 Enzyme folding with chemical denaturant…………….…………...…..13 2.8 Atomic absorption analysis…………….…………………………….………13 Result...……………………………………………………………….……….….…14 3.1 Protein expression and purification……………………………………….…15 3.2 Structure determination……………………………………………………...15 3.3 Overall structure.………….…….……….……….……….……….………...16 3.4 A metal ion-binding site……………………………………………………...17 3.5 Comparison with the pQC structure…………………………………………18 3.6 The active site structure……………………………………………………...20 3.7 Enzyme activity assay and kinetic assay…………………………………….20 3.8 Modeling and the catalytic mechanism of type I QCs……………………….22 3.9 Tests for resistance to chemical, acid, and thermal denaturation………….…22 Discussion…………...................................................................................................24 References…………………………………………………………………………...30 Figures……………………………………………………………………………….36 Tables………………………………………………………………………………..61 Poster………………………………………………………………………………..68 | |
| dc.language.iso | en | |
| dc.subject | 十字花科蔬菜黑腐病菌 | zh_TW |
| dc.subject | 麩酸胺環化酵素 | zh_TW |
| dc.subject | X光晶體結構 | zh_TW |
| dc.subject | 催化機制 | zh_TW |
| dc.subject | Xanthomonas campestris | en |
| dc.subject | glutaminyl cyclase | en |
| dc.subject | X-ray crystal structure | en |
| dc.subject | catalytic mechanism | en |
| dc.title | 第一個細菌麩酸胺環化酵素---十字花科蔬菜黑腐病菌麩酸胺環化酵素的表現、X光結構、催化機制、以及穩定性之研究 | zh_TW |
| dc.title | Expression, X-ray structure, catalytic mechanism, and stability of Xanthomonas campestris glutaminyl cyclase: the first bacterial glutaminyl cyclase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蕭傳鐙(Chwan-Deng Hsiao),馬徹(Alex Che-Ma),張崇毅(Chung-I Chang) | |
| dc.subject.keyword | 十字花科蔬菜黑腐病菌,麩酸胺環化酵素,X光晶體結構,催化機制, | zh_TW |
| dc.subject.keyword | Xanthomonas campestris,glutaminyl cyclase,X-ray crystal structure,catalytic mechanism, | en |
| dc.relation.page | 68 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-06-21 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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