利用定點突變探討胃幽門桿菌菌種26695之分枝酸變位酵素的活性區

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研究生：	林佳祺 Chia-Chi Lin
論文名稱：	利用定點突變探討胃幽門桿菌菌種26695之分枝酸變位酵素的活性區 Mutagenesis Study of Active Site Residues in Chorismate Mutase from Helicobacter pylori strain 26695
指導教授：	呂平江博士 Dr. Ping-Chiang Lyu
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2005
畢業學年度：	93
語文別：	英文
論文頁數：	53
中文關鍵詞：	分枝酸變位酵素、莽草酸途徑、胃幽門桿菌、定點突變、活性區
外文關鍵詞：	Chorismate mutase, shikimate pathway, Helicobacter pylori, mutagenesis, active-site residues
相關次數：	點閱：2 下載：0
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胃幽門桿菌26695是屬於革蘭氏陰性菌，一種具有鞭毛的螺旋狀桿菌。它寄居在人類的胃黏膜上，會造成胃潰瘍及胃癌的發生。由於胃幽門桿菌具有致病性，為了進一步地從染色體序列中得到更多的資訊，在1997年，Tomb等人發表了此菌的整個基因體序列。目前經由基因註解分析，預測HP0291的基因產物為分枝酸變位酵素 (chorismate mutase, CM)，它是合成芳香族胺基酸的莽草酸途徑 (shikimate pathway) 中的重要酵素，分枝酸變位酵素可以將分枝酸 (chorismate) 轉換成預苯酸 (prephenate)。根據分枝酸變位酵素的序列分析及蛋白質三級結構來作分類，HP0291是屬於分枝酸變位酵素中，具有單一功能的AroQ類型。在本研究中，利用大腸桿菌來表現胃幽門桿菌HP0291蛋白質，並利用鈷離子親和管柱加以純化。利用電腦來進行HP0291的胺基酸序列分析以及三維立體結構模擬，推測胺基酸序列中Arg13、Arg30、Lys41、Cys48、Arg51、Glu52、Ile55、Phe80、Ser83和Gln87，這些胺基酸殘基可能是位在分枝酸變位酵素的活性區中。在實驗方面，利用定點突變的方法得到14個突變株，透過生物物理與酵素活性的實驗，來探討這些胺基酸殘基在結構與功能上的關係。以旋光光譜儀 (circular dichroism, CD) 測試所得到的蛋白質的二級結構及熱穩定性發現，我們所做的定點突變並不會造成蛋白質結構或對溫度的穩定性產生明顯的改變，但卻對酵素的活性產生了大幅度的破壞。所以根據我們的實驗結果證實Arg13、Lys41、Cys48、Arg51、Glu52、Ile55、Phe80和Gln87，這些胺基酸殘基確實是對分枝酸變位酵素的活性具有影響，其中Ile55和Phe80兩個殘基在酵素活性上的貢獻，在過去的文獻中是沒有被報導過的。

Helicobacter pylori (H. pylori), a gram-negative, spiral-shaped and flagellated organism, colonized the gastric mucosa and significantly involved the cause of gastric ulcers and gastric cancers. The whole genome sequences of H. pylori strain 26695 have been reported in 1997. The HP0291 gene, encoding the chorismate mutase (CM), is a key enzyme of the shikimate pathway to the synthesis of aromatic amino acids. HP0291, which belongs to a monofunctional AroQ class of CMs, catalyzes the conversion of chorismate to prephenate. Based on the computational analysis of HP0291 protein sequence and 3-Dimensional structure, it suggests that Arg13, Arg30, Lys41, Cys48, Arg51, Glu52, Ile55, Phe80, Ser83 and Gln87 might be the active-site residues. Fourteen site-directed mutants of each residue have been constructed to test their structural and functional role. Although protein structure and stability of these mutants were similar to that of wild-type protein, most of the mutants showed 80-90% loss of their enzyme activities. Our results demonstrated that Arg13, Lys41, Cys48, Arg51, Glu52, Ile55, Phe80 and Gln87 were critical to the HP0291 functions.

Contents    1
Contents of Tables and Figures    2
Abstract    3
中文摘要    4
Abbreviations    5
Chapter 1. Introduction    6
1.1 Helicobacter pylori    6
1.2 Chorismate mutase    6
1.3 Structures of CM enzymes    8
1.4 Catalytic mechanism of CM enzymes    9
1.5 The theme of this thesis    10
Chapter 2. Materials & Methods    12
2.1 Materials    12
2.2 Strains, media, plasmids and growth conditions    12
2.3 Cloning of His-tagged HP0291    13
2.4 Site-directed mutagenesis of HP0291    14
2.5 Protein expression    15
2.6 Protein purification using AKTAprime device    16
2.7 ESI-MS analysis    17
2.8 Determination of protein concentration    17
2.9 Circular dichroism (CD) spectrometry    17
2.10 Chorismate mutase activity assay    19
Chapter 3. Results and Discussion    20
3.1 Computational analysis of HP0291 protein sequence and 3D structure    20
3.2 Targets for site-directed mutagenesis    21
3.3 Overexpression and purification of HP0291 variants    21
3.4 Biophysical analysis of substitution mutants    22
3.5 Critical active-site residues of HP0291    23
Chapter 4. Conclusion    25
Reference    27
Appendix 1    52
Appendix 2.    53


Contents of Tables and Figures



Table 1. E. coli strain and plasmids    33
Table 2. Sequence of oligonucleotide used for PCR and site-directed mutagenesis.    34
Table 3. The molecular weight of wild-type and mutant proteins    35
Table 4. Summary of the helicity and Tm of wild-type and mutant proteins    36
Table 5. Specific activities of wild-type and mutant proteins    37



Figure 1. Multiple sequence alignment of the five CM belongs to the AroQ
        family.    38
Figure 2. Intermolecular interactions between HP0291 and transition state analogue    39
Figure 3. Targets for site-directed mutagenesis.    40
Figure 4. Plasmid construction.    41
Figure 5. Expression of wild-type and mutant proteins.    42
Figure 6. SDS-PAGE of purified wild-type and mutant proteins.    45
Figure 7. SDS-PAGE of HP0291R30A.    47
Figure 8. Mass spectra of HP0291 wild-type and mutant proteins.    48
Figure 9. Far-UV circular dichroism (CD) spectra of wild-type and selected
        mutant proteins at 25℃.    50
Figure 10. Thermal-dependent unfolding (denaturation) curves of wild-type and mutant proteins.    51

                                

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