硫化屬古生菌中的酮醇酸還原異構酶結構分析

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姓名

汪致霖(Chih-lin Wang) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

硫化屬古生菌中的酮醇酸還原異構酶結構分析
(Structural analysis of ketol-acid reductoisomerase from Sulfolobus solfataricus)

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摘要(中)

酮醇酸還原異構酶可以在植物、真菌和細菌體內找到，而動物體內則沒有此酵素存在。酮醇酸還原異構酶可以應用在產生支鏈形胺基酸、生質能源以及除草劑。酮醇酸還原異構酶是支鏈形胺基酸生合成反應第二個反應的酵素，可以將乙酰乳酸轉變成2,3-二羥基異戊酸。此轉變過程包含了兩個反應，先依靠鎂離子將烷基轉移，之後在消耗菸鹼醯胺腺嘌呤二核苷酸磷酸(NADPH)還原兩個酮基。我們發現硫化屬古生菌(Sulfolobus solfataricus)中具有三個酮醇酸還原異構酶，分別是酮醇酸還原異構酶-1、酮醇酸還原異構酶-2和酮醇酸還原異構酶-3。這三個酮醇酸還原異構酶的基因序列之間並不完全相同，但是具有相同屬於酮醇酸還原異構酶的保守基因，且尚未了解其在生物體內分別的作用。在我的研究中，以大腸桿菌BL21(DE3)來大量表現酮醇酸還原異構酶-3，並利用快速蛋白質液相層析系統純化此蛋白質，我們發現酮醇酸還原異構酶-3在溶液中是以十二聚體的形式存在，並成功得到此十二聚體蛋白質的晶體，但晶體繞射訊號不佳。因此我們從電顯影像中，利用單分子重建法得到此十二聚體的三維平均結構，並用模擬的原子結構模型代入三維平均結構，嘗試以此方法來預測其四級結構組成，我們發現以單分子三維重構法重建出的酮醇酸還原異構酶-3結構與同一類的酮醇酸還原異構酶非常相似，在未來我們希望利用這個模擬的原子結構來探究其功能。

摘要(英)

Ketol-acid reductoisomerase (KARI) enzyme which is found in plants, fungi and bacteria but not in animals has attracted much interest for production of amino acids, biofuels and development of antimicrobial agents. The KARI is the second enzyme in the branched-chain amino acid biosynthesis pathway, which conversion of 2-acetolactate into 2,3-dihydroxyisovalerate. The conversion involves an Mg2+-dependent alkyl migration followed by a Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the 2-keto group. We have found that there are three homologs, IlvC1, IlvC2 and IlvC3 from sulfolobus solfataricus. The amino acid sequences of these three KARIs possess conserved regions despite they are not highly similar to one another and their function in vivo is not clear. In my study, ilvc3 were overexpressed in E. coli BL21 (DE3) and purified by FPLC. The ilvc3 was a dodecamer in solution with molecular weight ~450 kDa. We successfully found the condition of crystallize, however the diffraction of ilvc3 crystal was poor. Therefore, we built a 3D map of Ilvc3 by single particle 3D reconstruction atomic model, which could help us to determine the quaternary structure. It was discovered that the modeling structure of ilvc3 was similar to the other KARI in different species. In future, we attempt to study the function by the modeling atomic structure.

關鍵字(中)

★ 酮醇酸還原異構酶
★ 硫化屬古生菌
★ 蛋白質結構

關鍵字(英)

★ Sulfolobus solfataricus
★ ketol-acid reductoisomerase
★ Structural analysis

論文目次

中文摘要 - 1 -
Abstract - 2 -
Table of Contents - 3 -
Chapter.1 introduction - 6 -
1.1 Background - 6 -
1.1.1 Ketol-acid reductoisomerase - 6 -
1.1.2 Sulfolobus solfataricus - 8 -
1.1.3 Structural biology - 9 -
1.2 Specific aims - 10 -
Chapter.2 Material & Method - 12 -
2.1 Cloning and construction - 12 -
2.1.1 Polymerase chain reaction (PCR) - 12 -
2.1.2 Agarose gel electrophoresis - 12 -
2.1.3 Restriction enzyme digestion - 13 -
2.1.4 Ligation - 13 -
2.1.5 Transformation - 13 -
2.1.6 Sequencing - 14 -
2.2 Protein expression - 15 -
2.2.1 Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction system - 15 -
2.2.2 Time course - 15 -
2.3 Total protein extraction - 15 -
2.4 Protein purification - 16 -
2.4.1 Heat treatment assay - 16 -
2.4.2 Fast protein liquid chromatography (FPLC) - 16 -
2.4.3 Sodium dodecyl sulfate poly-acrylamide-gel-electrophoresis (SDS-PAGE) - 17 -
2.4.4 Protein quantification - 17 -
2.5 Structural analysis - 18 -
2.5.1 Size-exclusion chromatography - 18 -
2.5.2 Crystallization - 19 -
2.5.3 Circular Dichroism (CD) Spectrum - 19 -
2.5.4 Electron microscopy (EM) - 20 -
2.5.5 Single particle reconstruction - 20 -
2.5.6 Modeling - 21 -
2.5.7 Fit in 3D maps - 22 -
Chapter.3 Result - 23 -
3.1 Construction of IlvC3 from Sulfolobus solfataricus with his-tag - 23 -
3.2 Protein expression of IlvC3 - 23 -
3.3 Heat treatment assay and protein purification of IlvC3 - 24 -
3.4 The oligomerization of IlvC3 - 24 -
3.5 Crystallization of IlvC3 dodecamer - 25 -
3.6 IlvC-3 3D reconstruction - 25 -
3.7 The atomic structure modeling of IlvC3 - 26 -
Chapter.4 Discussion - 27 -
References - 29 -
Ⅰ. Figure - 32 -
Figure 1 Branched amino acid biosynthesis pathway. - 32 -
Figure 2 Reaction catalyzed by KARI, reaction intermediate analogues. - 33 -
Figure 3 Phylogenetic Tree - 34 -
Figure 4 IlvC3 conserved domains - 35 -
Figure 5 Colony PCR. - 36 -
Figure 6 IlvC3 DNA and protein sequence - 37 -
Figure 7 Time course of recombinant KARI at 37 oC and 25 oC - 38 -
Figure 8 The purification of ilvC-3 by SDS-PAGE. - 39 -
Figure 9 Circular Dichroism Spectrum. - 40 -
Figure 10 Molecular weight analysis. - 41 -
Figure.11 Crystal images - 43 -
Figure.12 Single particle reconstruction - 44 -
Figure 13 Fitting modeling sequence to 3D reconstruction images. - 47 -
Figure 14 The structure of class I KARI from different species - 48 -
Ⅱ. Table - 49 -
Ⅱ. Appendix - 53 -
Appendix 1 Class I KARI structure overview - 53 -
Appendix 2 Specificity loops of selected KARIs - 54 -
Appendix 3 Engineered pathway for isobutanol production - 55 -

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指導教授

陳青諭(Chin-Yu Chen)

審核日期

2015-8-25

推文