博碩士論文 105821017 詳細資訊




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姓名 曾祥慶(Hsiang-Ching Tseng)  查詢紙本館藏   畢業系所 生命科學系
論文名稱
(Functional analysis of biotin protein ligases from various species)
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摘要(中) 生物素可作為輔酶並參與生物體內一系列的羧化及去羧化反應,因此這些使用生物素當輔酶的酵素在許多重要代謝途徑中扮演不可或缺的角色,例如脂肪及含侧鏈胺基酸合成。生物素是用共價鍵結的方式與其相對應蛋白質上的離胺酸 (lysine) 做結合,過程需藉由特殊的酵素來執行,這些酵素稱為biotin protein ligase (BPL)。被修飾的離胺酸幾乎都位在羧化酶 (carboxylases) 中一段高度保守的序列AMKM。由於受質及酵素皆呈現高度保守,因此生物素化可以跨物種作用,例如酵母菌、阿拉伯芥、人類的BPL1都能有效地取代大腸桿菌的BPL (稱為birA)突變株。然而我的實驗結果卻顯示大腸桿菌的BPL不能互補酵母菌BPL1的剔除株。為了解開背後所蘊藏的分子機制,我試圖用融合蛋白、點突變、biotin濃度、和酵素濃度來研究此一現象,不幸地是所有的策略都無法讓酵素改變為正向的表現型。除此之外,本研究還想藉著融合Aquifex aeolicus BirA的催化區段與酵母菌Arc1p的tRNA結合區段,建構出能夠修飾tRNA的酵素。雖然這個融合蛋白仍具有催化的活性,不過修飾tRNA的活性不甚理想,故目前我們正改進實驗的條件。另外,我們也分析十株在演化過程相距甚遠之酵母菌BPL的序列相似性,結果發現他們的序列相似性約莫35%-60%,接著我們測試它們的跨物種互補活性,結果只有四株酵母菌株的BPL1可以將生物素加到自己的Arc1p,且這四株酵母菌的BPL1也可將生物素加至S. cerevisiae的Arc1p;另外六株則不行將生物素加到自己的Arc1p,也不可以將生物素加到S. cerevisiae的Arc1p,這些結果將有利於我們進一步分析BPL1的受質專一性。
摘要(英)

Biotin can serve as a coenzyme for a distinct set of catalytic reactions. Those biotin-dependent enzymes catalyze key steps which are involved in metabolic pathway. The covalent attachment of biotin to a conserved lysine residue in its cognate apoproteins is mediated by a specific enzyme, called biotin protein ligase (BPL). The biotinylated lysine residue is almost invariably positioned in a consensus sequence, AMKM, within the carboxylases. As a result, biotinylation can occur across widely divergent species. For example, the BPL1 homologues of yeast, Arabidopsis, and human can efficiently complement an E. coli birA mutant. Despite that, we found that some bacterial BPL1 homologues can functionally substitute for yeast BPL1, while others cannot substitute under similar conditions. To advance understanding of the functional property of this group of enzymes, we tried several different approaches, including mutagenesis, domain swapping, and gene expression. Unfortunately, none of the approaches can make the BPL rescue the negative phenotype. In addition, we tried to construct a biotin-tRNA-modifying enzyme by fusing a promiscuous Aquifex aeolicus BirA mutant and the tRNA-binding domain of yeast Arc1p. The resultant fusion protein still retained the biotin protein ligase activity, but was somehow inactive in modifying tRNA with biotin. Moreover, we have cloned BPL1 genes from ten yeast species and analyzed their sequence similarities. As it turned out, these yeast BPL1s possess 35%-60% similarities. Most interestingly, only four of the ten yeast BPL1s could biotinylate their own Arc1ps, and could biotinylate S. cerevisiae Arc1p. The remaining six yeast BPL1s failed to biotinylate their own Arc1ps, and could not biotinylate S. cerevisiae Arc1p. Such a finding may help us further delineate the substrate specificity of BPL1.
關鍵字(中) ★ 生物素
★ 生物素化
★ 離胺酸
★ 蛋白質轉譯後修飾
關鍵字(英)
論文目次 摘要
Abstract
Aknowledgement
List of figures
List of tables
Appendixes

Chapter Ⅰ Introduction 1
1.1. Overview of a protein post-translational modification - biotinylation 1
1.1.1. Protein biotinylation 1
1.1.2. Biotin and its physiological role 1
1.1.3. Mechanism of protein biotinylation by biotin protein ligase (BPL) 2
1.1.4. The classification of BPL 3
1.2. The structural difference and diversity of the biotinylated proteins 4
1.2.1. The structural difference of the biotinylated proteins 4
1.2.2. The diversity of the biotinylated proteins 5
1.3.. The promiscuous biotinylation activity for protein labeling 5
1.4. Specific aims 6

Chapter Ⅱ Materials and Methods 7
2.1. Strains, plasmids, and culture media 7
2.1.1. Strains 7
2.1.2. Plasmids 17
2.1.3. Culture media 8
2.2. Preparation and transformation of E. coli competent cells 9
2.2.1. Preparation of E.coli competent cells 9
2.2.2. Transformation of E. coli competent cells 10
2.3. Preparation and transformation of yeast competent cells 10
2.3.1. Preparation of yeast competent cells 10
2.3.2. Transformation of yeast competent cells 11
2.4. Construction of various BPLs and biotinylated substrates 12
2.5. Complementation assay for the cytoplasmic function of various BPLs 12
2.6 Protein preparation 12
2.7. SDS-PAGE 13
2.8. Western blot analysis 14
2.9. Purification of 6xHis-tagged proteins 16
2.10. In vitro biotinylation assay 19
2.11. Streptavidin-based gel mobility shift assay 19

Chapter Ⅲ Results 20
3.1. Properties of BPL retrieved from different species 20
3.2. Cross-species rescue activities of BPLs retrieved from distantly related
organisms 20
3.3. Overexpression of EcBirA cannot rescue the yeast knockout strain 21
3.4. Effect of biotin concentration on the biotinylation activity of EcBirA 22
3.5. Switching the enzyme specificities between EcBirA and yeast BPL1 23
3.6. Not all bacterial BPLs containing a DNA binding domain failed to rescue the growth defect of the yeast knockout strain 23
3.7. Biotinylation of yeast carboxylase by EcBirA 24
3.8. Construction of a biotin tRNA ligase 25
3.9. Promiscuous biotinylation by BPL mutants 26
3.10. Expanding the substrate tolerance of BPL through exploration of BPL1 enzymes from various yeast species 26

Chapter Ⅳ Discussion 29
4.1. EcBirA cannot rescue the yeast BPL1 knockout strain 29
4.2. Construction of a biotin tRNA ligase 30
4.3. Screening for a BPL1 enzyme that can attach biotin analogues or derivaties to proteins 30

Bibliographies 32
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指導教授 王健家 審核日期 2017-6-21
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