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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 詹迺立(Nei-Li Chan) | |
dc.contributor.author | Yi-Ting Li | en |
dc.contributor.author | 李宜庭 | zh_TW |
dc.date.accessioned | 2022-11-25T08:03:07Z | - |
dc.date.copyright | 2021-09-16 | |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-08-03 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82939 | - |
dc.description.abstract | 細菌對抗生素產生抗藥性是現今公共衛生領域面臨的重大挑戰之一,因此,開發新的治療策略為一迫切的需求。改造現有的核苷類抗生素以產生新的抗生素,是對抗細菌耐藥性的方法之一。因此,詳細瞭解核苷類抗生素的生物合成機制有其必要性。多抗黴素(Polyoxin)是一種真菌核苷類似物,主要由核苷骨架、多肟酸(POIA)和氨甲醯多聚草氨酸 (CPOAA) 三部分組成。多抗黴素雙羥化酶 (PolL) 是參與氨甲醯多聚草氨酸 (CPOAA) 生合成途徑的重要蛋白,它催化 α-氨基-δ-氨基甲酰基羥基戊酸 (ACV) 的連續羥基化反應,首先在四號碳骨架上催化生成第一個羥基,接著在三號碳催化上產生第二個羥基,完成氨甲醯多聚草氨酸 (CPOAA)的生合成。電腦分析 (In silico analysis) 結果顯示,PolL之立體結構應為典型的桶狀折疊 (cupin fold),催化中心中包含具高度保留性、與二價鐵離子(Fe(II)) 結合的蛋白序列 HX(D/E)XnH。功能研究進一步顯示 PolL 需要氧氣 (O2) 和2-酮戊二酸 (ɑ-Ketoglutarate) 作為輔受質,以及二價鐵離子 Fe(II) 作為輔因子。基於這些特徵, 推測PolL應屬於非血基質鐵 (non-heme iron) /α-酮戊二酸 (α-ketoglutarate) 依賴型雙氧化酶蛋白家族 (2OGXs)。此外,此蛋白家族成員催化中心結構均具有由八股反平行的β-褶板摺疊之雙鏈 β-螺旋 (DSBH) 核心,內含由兩個組胺酸 (Histidine)與一個天門冬胺酸 (Aspartic acid) 或麩胺酸 (Glutamic acid)形成與二價鐵離子結合的模體。在催化過程中,2OGXs家族蛋白會利用具強氧化力的四價鐵離子Fe(IV)-oxo 中間物來催化各式反應。此外,目前已發現,若在受質上加上疊氮基 (azido group) 此輔助性官能基,則PolL 會由原先催化的連續羥基化反應轉變成催化非天然之腈基化反應 (nitrile installation),顯示出2OGXs家族蛋白催化反應的多樣性。因此,詳細的了解2OGXs家族蛋白的催化機制有助於將其用於潛在的生物催化劑。 為了探索PolL如何達成立體特異性且連續之羥基化反應,本研究的首要目標是解析PolL的立體結構,以探討其催化反應的詳細機制。來自可可鏈黴菌的PolL基因已被建構於表達質體,且重組 PolL 蛋白可以成功地於大腸桿菌中表達並純化至均質。然而,純化的 PolL 在室溫且高蛋白濃度條件下並不穩定,並且在結晶篩選過程中極容易形成嚴重沉澱,且僅有球晶 (spherulites) 生成。雖然經過微調後得到晶體,但X光繞射實驗結果顯示是鹽類晶體。為了得到蛋白晶體,我們進行了緩衝溶液篩選 (buffer screen) 以尋找更合適的緩衝溶液條件,提升蛋白穩定性以促進蛋白結晶,篩選結果顯示 PolL 在磷酸緩衝溶液中展現更好的穩定性。然而,儘管 PolL 在掃晶實驗過程中沈澱情形大幅下降,但僅生成了鹽類晶體,而若將磷酸緩衝溶液濃度調降,則此時蛋白的穩定度便顯著下降,在掃晶過程當中形成較嚴重的沈澱情形。 18-冠醚-6 (18-crown-6-ether) 是一種環狀醚,會通過穩定帶正電荷氨基酸的側鏈構形來調節蛋白質表面亂度以助蛋白結晶,因此也被添加到 PolL 中。儘管添加了18-冠醚-6 的蛋白樣品確實表現出更高的穩定性,蛋白樣本能濃縮至更高的濃度 (10 mg/mL),在掃晶實驗中沈澱的情況也較以往輕微。然而,於此同時,樣本變得非常粘稠,且生成的晶體經 X 光繞射分析顯示仍為鹽類。後又在新的結晶條件中發現球晶 (spherulites) 生成,由硬度判斷應是蛋白堆疊物,未來將可進行微調,以得到蛋白晶體。在原本的重組蛋白質體外,我們也嘗試不同的基因架構以幫助形成蛋白晶體。根據序列比對及二級結構預測的結果,我們建構一個N端截短的 PolL 蛋白以促進結晶,雖然此蛋白可以成功被大腸桿菌表達,但在純化過程中出現溶解度不佳的問題。於是,我們又再設計了另一個能夠降低的表面亂度的 PolL 突變質體 (PolL_SER),以增加我們獲得晶體的機會。目前 PolL_SER 蛋白已可成功被大腸桿菌表達,且可以成功被純化,日後將可持續搜尋PolL_SER 的結晶條件。 | zh_TW |
dc.description.provenance | Made available in DSpace on 2022-11-25T08:03:07Z (GMT). No. of bitstreams: 1 U0001-2507202117060900.pdf: 40985975 bytes, checksum: 012166b364774f0c1ae66c8fb1c4c6c0 (MD5) Previous issue date: 2021 | en |
dc.description.tableofcontents | "Contents 口委審定書 II 致謝 III 摘要 IV Abstract VI Contents IX List of Figures XIII List of Tables XV 1. Introduction 1 1.1 Polyoxin dihydroxylase: an enzyme catalyzing a rare sequential hydroxylation in the biosynthesis of CPOAA, a component of polyoxin. 2 1.2 PolL belongs to the non-heme Fe(II)-2-oxoglutarate-dependent dioxygenases superfamily 6 1.3 The consensus catalytic mechanism of substrate hydroxylation operated by the non-heme Fe(II)/2OG-dependent dioxygenases 9 1.4 A proposed mechanism of PolL-catalyzed nitrile installation reactions 11 1.5 Specific aims of this thesis 12 2. Materials and Methods 14 2.1 Protein expression systems 15 2.1.1 DNA construct 15 (1) First recombinant DNA construct 15 (2) N-terminal truncated DNA construct 16 (3) Surface Entropy Reduction DNA construct 17 2.1.2 Small-scale expression test 18 (1) pET28a-His6-PolL and pET28a-His6-PolL K105A/E107A/E108A 18 (2) pET21b-PolL_dN8 20 2.1.3 Large-scale protein expression 20 2.2 Protein purification of PolL and SER variant 21 2.2.1 Cell lysis 21 2.2.2 Liquid chromatography 21 (1) Immobilized metal affinity chromatography, IMAC 21 (2) Cation-exchange chromatography, CIEC 22 (3) Size exclusion chromatography, SEC 23 2.3 Protein crystallization 23 2.3.1 Sample preparation 23 2.3.2 Crystallization screen 24 2.3.3 Crystallization reagent optimization 25 2.3.4 Fluorescence-based Thermal shift assay 26 3. Results 27 3.1. Expression and purification of N-terminal hexa-histidine tagged PolL 28 3.2. Crystallization and preliminary diffraction analysis of apo PolL 29 3.3. Optimization of PolL storage buffer by thermal shift assay 30 3.4. 18-crown-6 ether to improve protein stability 32 3.5. N-terminal truncated PolL was less soluble and could not be purified 33 3.6. Surface entropy reduction was applied to enhance crystallizability for PolL 34 4. Discussion 36 4.1. The first recombinant N-terminal His-tagged PolL 37 4.2. Thermal shift assay of PolL 38 4.3. 18-Crown-6 ether for PoL crystallization 39 4.4. N-terminal truncated PolL 40 4.5. Surface entropy reduction mutagenesis of PolL 40 4.6. Future works 41 5. Figures 42 6. Tables 68 7. Reference 79 8. Appendix 88 List of Figures Figure 1-1. The structure of polyoxin complex and UDP-N-acetylglucosamine 4 Figure 1-2. The chemical structure of polyoxin 5 Figure 1-3. The CPOAA biosynthetic pathway 6 Figure 1-4. Illustration of reactions catalyzed by the Fe/2OG enzyme superfamily. 8 Figure 1-5. A close-up view of the active site of a Fe/2OG enzyme 8 Figure 1-6. The consensus catalytic strategy of substrate hydroxylation shared among non-heme iron(II)/2-OG dependent dioxygenases 11 Figure 1-7. The brief canonical hydroxylation pathway of 2OGX and the possible mechanism of non-native nitrile installation catalyzed by PolL. 12 Figure 1. Expression test for N-terminal His-tagged PolL. 43 Figure 2. IMAC purification of native PolL. 44 Figure 3. Mono S purification of native PolL. 46 Figure 4. Superdex 200 pg purification of native PolL. 48 Figure 5. Spherulites and crystals found in Index 48 50 Figure 6. Thermal shift assay of PolL and preliminary X-ray analysis of crystals obtained from phosphate-containing buffer. 51 Figure 7. Preliminary X-ray analysis of crystals obtained from a 18-crown-6-ether added PolL in phosphate-containing buffer. 53 Figure 8. Spherulites found in Morpheus 1-34 of the PolL•Fe•2OG•18-crown-6-ether protein sample purified by Tris buffer were likely resulted from odered protein packing. 54 Figure 9. Sequence alignment and protein secondary structure prediction of PolL 55 Figure 10. Expression test for PolL_dN8 58 Figure 11. Nickel column purification of PolL_dN8. 59 Figure 12. Expression test for PolL_SER mutant 61 Figure 13. Nickel column purification of PolL_SER. 63 Figure 14. Purification of PolL_SER by ion-exchange and gel-filtration chromatography. 65 Figure 15. Mass spectrometry and structure modeling of PolL_SER. 67 List of Tables Table 1-1. Strains used in this study. 69 Table 1-2. Plasmids used in this study. 69 Table 2-1. Mediums used for E. coli culture. 71 Table 2-2. Antibiotic usage of E. coli culture. 71 Table 3-1. Buffers and their components used for native PolL purification. 72 Table 3-2. Buffers and their components used for N-terminal truncated PolL (PolL_dN8) purification. 73 Table 3-3. Buffers and their components used for PolL_SER purification. 73 Table 4. The screen conditions of thermal Shift assay 75 Table 5-1. Sequence alignment used for N-terminal truncated protein engineering 76 Table 5-2. Proposed SER mutations predicted by SERp server 76 Table 6-1. Reagents of commercial kits which have produced salt crystals which already checked by the X-ray diffraction test. 77 Table 6-2. Reagents of commercial kits which have produced spherulites checked by the crush test. 78" | |
dc.language.iso | en | |
dc.title | 探討多抗黴素雙羥化酶催化連續羥基化及非天然腈基化之結構機轉 | zh_TW |
dc.title | Structural study of polyoxin dihydroxylase from Streptomyces cacaoi | en |
dc.date.schoolyear | 109-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 曾秀如(Hsin-Tsai Liu),徐駿森(Chih-Yang Tseng) | |
dc.subject.keyword | 非血基質鐵/α-酮戊二酸依賴型雙氧化酶,多抗黴素雙羥基化酶,連續羥基化反應,非天然腈基化, | zh_TW |
dc.subject.keyword | non-heme FeII/2-oxoglutarate-dependent dioxygenase,polyoxin dihydroxylase,sequential hydroxylation,nitrile installation, | en |
dc.relation.page | 91 | |
dc.identifier.doi | 10.6342/NTU202101724 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2021-08-04 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
dc.date.embargo-lift | 2023-08-03 | - |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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