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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王惠鈞 | |
dc.contributor.author | Ko-Mei Chen | en |
dc.contributor.author | 陳可玫 | zh_TW |
dc.date.accessioned | 2021-06-08T04:22:21Z | - |
dc.date.copyright | 2010-07-28 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-07-05 | |
dc.identifier.citation | REFERENCES
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22613 | - |
dc.description.abstract | Part A.
脂肪酶是一種水溶性酶,可催化不溶於水之脂質類受質的水解。如同大多數水解酶,脂肪酶的Ser-His-Asp活性中心類似於許多已知的絲氨酸蛋白酶。並且, 脂肪酶都有一個α/β水解酶結構。近年來脂肪酶備受關注,是因為其潛在的工業應用價值。我們成功的解出了新型脂肪酶: 嗜超高溫古生菌Archaeoglobus fulgidus脂肪酶(AFL)的晶體結構,並已確定為約 1.8的解析度。這種酶的最佳活性溫度在70-90攝氏度的範圍內,最佳活性酸鹼值則在pH值10-11。AFL是由一個 N端α/β-水解酶結構,一個蓋子結構,和C端beta-barrel結構所組成。而它N端的催化區域則是6個β-sheet,兩側被7個α-helix所包圍,4個在一面,3個在另一面。AFL的C端脂質結合區域是由14個β-sheet所組成,其上端有一個區域覆蓋著底下高度疏水的受質結合點。AFL的3個活性催化重要胺基酸(Ser136,Asp163和His210)和氧離子洞(Leu31和Met137)的位置類似於其他脂肪酶。長鏈受質坐落在這C端和N端兩個領域的中間。將AFL和另一相似的Bacillus subtilis脂肪酶之催化結構區域做比較會發現受質與它們的結合是採用相反的方向。由於AFL的C端區域存在一個很大的疏水環境使得它對長鏈脂肪酸受質能夠有較高的活性。在兩個區域之間非常大的相互作用表面積可能有助於AFL的熱穩定性。Asp 61 和 Lys101被確定為可以調控蓋子區域的兩個重要氨基酸。此兩個氨基酸的氫鍵模式是根據pH來改變,這也許可以解釋為何此脂肪酶在鹼性pH值仍有很高的活性。我們定義C端β-barrel區域的角色為受質結合區,並且可以提供結構穩定的力量。如對此脂肪酶的嗜鹼性高溫與穩定性做進一步的改良工程將可幫助大幅提升脂肪酶的應用價值。 Part B. 四異戊二烯焦磷酸合成酶(GGPPS)催化法尼基焦磷酸(FPP)與異戊烯基焦磷酸(IPP)生成20個碳的焦磷酸(GGPP),焦磷酸是一個胡蘿蔔素,葉綠素,geranylgeranylated蛋白質,脂類,也是與古細菌聯接的脂質的前驅物。我們成功解出雙磷酸鹽藥物(Bisphosphonate)跟抗癌標的四異戊二烯焦磷酸合成酶的複合結構,雙磷酸鹽藥物平常使用於治療骨質疏鬆症引起的骨吸收和腫瘤引起的高鈣血症,它同時也是法尼基焦磷酸合酶(FPPS)的強效抑製劑。在後來的研究發現到雙磷酸鹽藥物也能抑制GGPPS的活性。我們也發現不分枝的雙磷酸鹽藥物可能會跟受質或產物的位置分別或同時形成鍵結,有一些V型的雙磷酸鹽藥物甚至會與四異戊二烯焦磷酸合成酶(GGPPS)的受質與產物位置都形成鍵結,並且會對於GGPPS有抑制效果。雙磷酸鹽藥物也同時會與三個鎂離子形成鍵結,其位置就與在法呢基焦磷酸合成酶(FPPS)結構中所見雷同。在我們得到的七個結構中,我們發現每個分子中有多達 4個結合位點。在此結果中發現一些GGPPS抑製劑可以跟受質或產物的位置分別或同時形成鍵結,相信此研究中所得到的雙磷酸鹽藥物跟抗癌標的四異戊二烯焦磷酸合成酶的複合結構與其抑制效果的分析將會有助於癌症藥物的研發與設計。 | zh_TW |
dc.description.abstract | Part A.
Lipase is a water-soluble enzyme that catalyzes the hydrolysis of ester bonds in water-insoluble lipid substrates. As most carboxylic ester hydrolases, Lipases contain a Ser-His-Asp catalytic triad similar to the much studied serine proteases. They also share a common structural framework alpha/beta hydrolase fold. In recent years lipases have attracted much interest because of their potential use in industrial applications. Several crystal structures of AFL, a novel lipase from the archaeon Archaeoglobus fulgidus, complexed with various ligands, have been determined at about 1.8 Å resolution. This enzyme has optimal activity in the temperature range of 70-90 ℃ and pH 10-11. AFL consists of an N-terminal alpha/beta-hydrolase fold domain, a small lid domain, and a C-terminal beta-barrel domain. The N-terminal catalytic domain consists of a 6-stranded beta-sheet flanked by seven alpha-helices, four on one side and three on the other side. The C-terminal lipid binding domain consists of a beta-sheet of 14 strands and a substrate covering motif on top of the highly hydrophobic substrate binding site. The catalytic triad residues (Ser136, Asp163, and His210) and the residues forming the oxyanion hole (Leu31 and Met137) are in positions similar to those of other lipases. Long-chain lipid is located across the two domains in the AFL-substrate complex. Structural comparison of the catalytic domain of AFL with a homologous lipase from Bacillus subtilis reveals an opposite substrate binding orientation in the two enzymes. The presence of a large hydrophobic tunnel in the C-terminal domain in AFL enables it to have a higher preference toward long-chain substrates. The unusually large interacting surface area between the two domains may contribute to thermostability of the enzyme. Two amino acids, Asp 61 and Lys101, are identified as hinge residues regulating movement of the lid domain. The hydrogen-bonding pattern associated with these two residues is pH dependent, which may account for the optimal enzyme activity at high pH. We defined the role of the C-terminal β-barrel domain of the AFL as an anchoring domain for its substrate as well as providing the stability force. Further engineering of this novel lipase with high temperature and alkaline stability will find its use in industrial applications. Part B. Geranylgeranyl pyrophosphate synthase (GGPPS) catalyzes a condensation reaction of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C20 geranylgeranyl pyrophosphate (GGPP), which is a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether linked lipids. We report several complexed structures of geranylgeranyl pyrophosphate synthase, a target for anticancer drugs. Bisphosphonate drugs used for osteoclast-mediated bone resorption and tumor-induced hypercalcemia are potent inhibitors of farnesyl pyrophosphate synthase (FPPS). Bisphosphonate drugs were shown to inhibit the activity of GGPPS. Bisphosphonates containing unbranched side chains bind to either the farnesyl diphosphate (FPP) substrate site or the geranylgeranyl diphosphate (GGPP) product site, and in one case, both sites, with the bisphosphonate moiety interacting with 3 Mg (2+) that occupy the same position as found in FPPS. However, each of three 'V-shaped' bisphosphonates bind to both the FPP and GGPP sites. In each of the seven structures investigated, we found that there were up to four binding sites per monomer. These results show that some GGPPS inhibitors can occupy both substrate and product site and that binding modes as well as activity can be accurately predicted, facilitating the further development of GGPPS inhibitors as anticancer agents. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T04:22:21Z (GMT). No. of bitstreams: 1 ntu-99-F93b46036-1.pdf: 13729495 bytes, checksum: a352f6141e681f1a62a50122af3c4bf6 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 目 錄
誌謝.....................................................i 中文摘要Part A...........................................ii 中文摘要Part B..........................................iii Abstract Part A.........................................iv Abstract Part B.........................................vi Part A. Structure of the alkalohyperthermophilic Archaeoglobus fulgidus lipase contains a unique c-terminal domain essential for long-chain substrate binding 1. INTRODUCTION...........................................2 2. MATERIAL AND METHOD....................................7 2.1 Materials.............................................7 2.2 Protein expression and purification of the truncated, native, and mutant AFL...................................7 2.3 Crystallization and data collection for AFL..........9 2.4 Structure determination and refinement..............10 2.5 Site-directed mutagenesis of AFL.....................11 2.6 Construction of the C-terminal truncated mutant.....12 2.7 Enzyme assay........................................12 2.8 Interfacial activation assay........................12 2.9 PDB accession numbers...............................13 3. RESULTS...............................................13 3.1 X-ray structure determination........................13 3.2 Overall structure of AFL.............................15 3.3 Comparison with Bacillus subtilis lipase (BSL).......17 3.4 Active site..........................................17 3.5 Acyl–binding site...................................18 3.6 The domain-domain interface..........................21 3.7 AFL is a true lipase that contains a unique C-domain structure for substrate binding and catalysis...........22 4. DISCUSSION............................................25 LIST OF TABLES Part A Table 1. Data collection and refinement statistics for AFL crystals................................................28 Table 2. AFL heavy-atom derivatives and MIR statistics...............................................29 Table 3. The hydrophobic interaction between the C-terminal domain and the N-terminal domain...............30 LIST OF FIGURES Part A Fig. 1 The catalytic action of lipases...................32 Fig. 2 Reaction mechanism of lipases.....................33 Fig. 3 The construct of A. fulgidus lipase...............34 Fig. 4 The purification steps of A. fulgidus lipase......35 Fig. 5 Crystal of AFL....................................36 Fig. 6 Superimposition of the subunits of AFL............37 Fig. 7 The native structure of AFL with ion binding......38 Fig. 8 The ion binding sites in AFL......................39 Fig. 9 A topology diagram corresponding to each domain...40 Fig. 10 The overall structure of AFL.....................41 Fig. 11 The substrate used for the activity test.........42 Fig. 12 The overall structure of B. subtilis lipase......43 Fig. 13 A stereo view of the superimposition of the AFL structure with BSL.......................................44 Fig. 14 Structure-based sequence alignment of AFL and BSL......................................................45 Fig. 15 A stereo view of the superimposition of the catalytic region of AFL and BSL..........................46 Fig. 16 Details of the hydrophobic tunnel of AFL......................................................47 Fig. 17 The pH-dependent conformational change of the hinge residues...........................................48 Fig. 18 The substrate binding site of AFL and BSL........49 Fig. 19 The domain-domain interface......................50 Fig. 20 Details of the interaction between the domain-domain interface.........................................51 Fig. 21 Interfacial activation effect of AFL.............52 Fig. 22 Substrate specificity of recombinant AFL mAFL and tAFL.....................................................53 Fig. 23 Optimum temperature, thermostability, optimum pH, and pH-stability assay of recombinant AFL................54 Part B. Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates: a crystallographic investigation 1. INTRODUCTION..........................................56 2. MATERIAL AND METHOD...................................60 2.1 Chemicals...........................................60 2.2 Protein Expression and Purification. ................60 2.3 Crystallization and data collection for GGPPS complexes................................................62 2.4 Structure determination and refinement...............64 2.5 GGPPS inhibition.....................................64 3. RESULTS..............................................66 4. DISCUSSION............................................71 LIST OF TABLES PART B Table 1. Data Collection and Refinement Statistics for GGPPS-Bisphosphonate Crystals............................74 Table 2. Data collection and refinement statistics for GGPPS-bisphosphonate crystals............................75 Table 3. Data collection and refinement statistics for GGPPS-bisphosphonate crystals............................76 Table 4. GGPPS inhibition result.........................77 LIST OF FIGURES PART B Fig. 1 Synthesis of linear all trans-isoprenyl pyrophosphates and trans,cis-isoprenyl pyrophosphates...........................................79 Fig. 2 Reactions of (trans) prenyl diphosphate synthases in isoprenoid biosynthesis.............................................80 Fig. 3 Sequence alignment of trans-prenyl-transferases...81 Fig. 4 Biosynthesis of isoprenoids and proposed mechanisms for trans- prenyltransferases............................82 Fig. 5 GGPP biosynthesis pathway.........................83 Fig. 6 Backbone chemical structure of a bisphosphonate...84 Fig. 7 Bisphosphonate compounds investgated in the previous experiments.....................................85 Fig. 8 Crystallization condition.........................86 Fig. 9 Binding modes observed in S. cerevisae GGPPS-bisphosphonate complexes.................................87 Fig. 10 Investigated bisphosphonate compounds............88 Fig. 11 The 2Fo-Fc electron density maps.................89 Fig. 12 GGPPS dimer structure and binding motifs.........90 Fig. 13 The binding motifs of several inhibitors.........91 Fig. 14 11 (BPH-23) bound to GGPPS superimposed on FPP, FsPP + IPP and GGPP complex structures...................92 Fig. 15 GGPPS dimer structure and binding motifs of branched bisphosphonates.................................93 Fig. 16 Binding motif of bisphosphonate-GGPP complex structures. Compound 10..................................94 Fig. 17 Compound 9 bound to GGPPS superimposed on FsPP + IPP and GGPP complex structures..........................95 REFERENCES...............................................96 LIST OF PUBLICATION....................................102 | |
dc.language.iso | en | |
dc.title | A. 脂肪酶的C端之結構對於較長鏈受質的結合之重要性
B. 雙磷酸鹽藥物對於四異戊二烯焦磷酸合成酶的抑制效果在結構上的分析 | zh_TW |
dc.title | A. Structure of the Alkalohyperthermophilic Archaeoglobus fulgidus Lipase Contains a Unique C-Terminal Domain Essential for Long-Chain Substrate Binding
B. Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates: a crystallographic investigation | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 詹迺立,馬徹,李冠群,陳光超 | |
dc.subject.keyword | 蓋,催化區域,界面,熱穩定性,脂肪酶,雙磷酸鹽藥物,抗癌,四異戊二烯焦磷酸合成酶,法呢基焦磷酸,晶體學, | zh_TW |
dc.subject.keyword | lid,catalytic site,interface,thermostability,lipase,bisphosphonate,anticancer,geranylgeranyl diphosphate synthase,Farnesyl diphosphate,crystallographically, | en |
dc.relation.page | 103 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2010-07-06 | |
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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