以結構及酵素活性之方法研究胃幽門螺旋菌及結核分支桿菌之莽草酸途徑酵素：Shikimate Dehydrogenase 及 3-Dehydroquinate Synthase

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研究生：	陳粲然 Chen, Tsan-Jan
論文名稱：	以結構及酵素活性之方法研究胃幽門螺旋菌及結核分支桿菌之莽草酸途徑酵素：Shikimate Dehydrogenase 及 3-Dehydroquinate Synthase Structure-activity Investigation of Shikimate Pathway Enzymes from Helicobacter pylori and Mycobacterium tuberculosis : Shikimate Dehydrogenase and 3-Dehydroquinate Synthase
指導教授：	王雯靜
口試委員:	許宗雄許銘華楊進木
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	89
中文關鍵詞：	胃幽門螺旋菌、結核分支桿菌、莽草酸、晶體結構
外文關鍵詞：	Shikimate
相關次數：	點閱：3 下載：0
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　　莽草酸代謝途徑 (shikimate pathway) 對於微生物、植物及寄生蟲類是不可獲缺的，其最終產物 chorismate 是合成芳香族胺基酸的前驅物，如酪胺酸及苯丙胺酸。由於莽草酸途徑並不存在於哺乳動物當中，固此途徑的酵素就成為了發展抗微生物藥物的良好標的。其中 shikimate dehydrogenase (SDH) 是此代謝途徑七個酵素中的第四個，負責催化 shikimate 轉換為 3-dehydroshikimate，在本實驗室的前期研究中已經解出胃幽門螺旋菌 (Helicobacter pylori, Hp) 之 SDH 的晶體結構，並成功篩選出數種具有抑制效果的化合物。本研究延續前期實驗的結果，從結構以及生化分析的角度探討抑制效果較明顯的 NSC408168 對於 HpSDH 抑制的分子作用機制為何，首先藉由定點突變的方式建構了六個 HpSDH 突變株 (Y210A, Y210S, Y210F, Q237A, Q237N, 及 Q237K)，並成功解出其中五個的晶體結構，另外由 iTC 的實驗發現六個突變株對 NSC408168 的結合能力均有所下降。此外，本研究中也解出由抗生素臨床治療失敗的病人身上所分離出來的 Hp 菌株之 SDH 結構 (HpSDH-v2356)，其胺基酸序列與野生型Hp有93%的相似度，並且擁有較高的結構變異性，其酵素反應活性也優於野生型酵素，有趣的是，在IC50 的測試中也發現 NSC408168 對於此臨床分離株也有較好的抑制效果。深入探討 HpSDH 突變株與臨床分離菌株在結構以及序列上的差異將有助於瞭解 NSC408168 的作用機制，並可作為未來發展更有效抑制藥物的研究基礎。
　　另一方面，3-dehydroquinate synthase (DHQS) 是莽草酸途徑中的第二個酵素，以NAD+作為輔因子，將 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) 催化轉換成 3-dehydroquinate，本研究藉由分子選殖的方法表現出結核分枝桿菌 (Mycobacterium tuberculosis, Mt) 之 DHQS 蛋白，並且成功解析出 NAD+ 共結晶以及 DAHP 共結晶的晶體結構，解析度分別到達 2.47 Å 及 1.80 Å，為目前各物種之 DHQS 蛋白中，第一個解析出來的 DAHP 共結晶結構，兩個結構互相比較可發現其 domain 開合程度略有差異，一個可視為 open form，一個為 close form。藉由仔細分析這兩個高解析度結構，將可進一步瞭解其酵素催化機制，並提供未來針對 DHQS 進行抑制物篩選以及電腦輔助藥物設計的參考。

Shikimate pathway is an essential synthetic pathway for microbes and parasites but absent in mammals. The end product, chorismate, serves as a precursor for the synthesis of aromatic amino acids, including tyrosine and phenylalanine. The importance of shikimate pathway makes it a potential antimicrobial target for new drug discovery. Shikimate dehydrogenase (SDH) is the fourth enzyme involved in the shikimate pathway, which catalyzes the oxidation of shikimate to 3-dehydroshikimate. The structure of shikimate dehydrogenase from Helicobacter pylori had been determined in previous studies and several potential inhibitors had been identified, including NSC408168. Here, we focused on the study of inhibitor NSC408168 toward our target enzyme by performing site-directed mutagenesis. Five HpSDH mutant structures (Y210A, Y210S, Q237A, Q237N, and Q237K) had been solved and they showed subtle differences compared with the wild type structure. The isothermal titration calorimetry analysis suggested that the HpSDH mutants had weaker binding affinities with NSC408168 if compared to the wild type. In addition, a structure of SDH from a clinical isolated H. pylori strain v2356, which retained 93% sequence identity with wild type HpSDH, had been solved in this study and it showed higher structure flexibility and enzyme activity compared to the wild type structure. Surprisingly, NSC408168 showed better inhibition toward HpSDH-v2356 with the IC50 value of 0.82 μM. Together, these results may provide clues for the inhibition mechanism of NSC408168 toward HpSDH and serve as a template for designing more effective inhibitors in the future.
On the other hand, 3-dehydroquinate synthase (DHQS) involved in the second step of shikimate pathway, which catalyzes the conversion of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) to 3-dehydroquinate, by using NAD+ as a cofactor. In this study, we had solved the complex structures of DHQS from Mycobacterium tuberculosis with the cofactor (NAD+) and substrate (DAHP), at a resolution of 2.47 Å and 1.80 Å, respectively. This is also the first DAHP-complexed structure of DHQS being discovered among different species. The two structures show an open form and close form transition. The high resolution of MtDHQS structures provide us an insight in the catalytic mechanism and potential inhibitors can be identified for antimicrobial treatment.

中文摘要 …………………………………………………………………………………………  i
Abstract  ………………………………………………………………………………………… ii
誌謝 ………………………………………………………………………………………………iii
Content  ……………………………………………………………………………………………v
List of Figures ……………………………………………………………………………………viii
List of Tables ………………………………………………………………………………………x
 Introduction  ………………………………………………………………………………… 1
1  Helicobacter pylori  ………………………………………………………………………  1
2  The epidemiology and treatment of Helicobacter pylori …………………………………  1
3  Mycobacterium tuberculosis ………………………………………………………………  2
4  The epidemiology and treatment of Mycobacterium tuberculosis  ………………………  2
5  Shikimate pathway  ………………………………………………………………………  3
6  3-Dehydroquinate synthase ………………………………………………………………… 3
7  Preliminary results on MtDHQS …………………………………………………………… 3
8  Shikimate dehydrogenase  ………………………………………………………………… 4
9  Preliminary results on HpSDH ……………………………………………………………… 4
10  Purpose and specific aims ………………………………………………………………… 5
 Material and Methods ………………………………………………………………………  6
1  HpSDH cloning and site-directed mutagenesis ……………………………………………  6
2  HpSDH expression, purification and condensation  ……………………………………… 7
3  Determination of HpSDH relative enzyme activity  ……………………………………… 8
4  Isothermal titration calorimetry ……………………………………………………………  8
5  Determination of NSC408168 IC50 toward HpSDH ………………………………………  8
6  HpSDH crystallization screening …………………………………………………………… 9
7  Optimization of HpSDH crystallization conditions ………………………………………… 9
8  X-ray diffraction and data processing  …………………………………………………… 10
9  HpSDH model building and refinement  ………………………………………………… 10
10  Sequence alignment, structure validation and structure comparison  …………………… 11
11  Structural representation ………………………………………………………………… 11
12  Material and methods for MtDHQS study ……………………………………………… 11
12.1  MtDHQS cloning  …………………………………………………………………… 11
12.2  MtDHQS protein expression and purification  ……………………………………… 12
12.3  MtDHQS crystallization ……………………………………………………………… 12
12.4  MtDHQS protein model construction  ……………………………………………… 13
 Results  ……………………………………………………………………………………… 14
1  HpSDH gene cloning ……………………………………………………………………… 14
2  HpSDH expression and purification ……………………………………………………… 14
3  Relative enzyme activity of HpSDHs  …………………………………………………… 14
4  Thermal dynamic analysis of NSC408168 binding with HpSDHs  ……………………… 15
5  IC50 of NSC408168 toward different HpSDHs …………………………………………… 15
6  HpSDH crystallization  …………………………………………………………………… 15
6.1  Crystallization of HpSDH mutants  …………………………………………………… 15
6.2  Crystallization of HpSDH clinical variant  …………………………………………… 16
7  X-ray diffraction data collection and processing  ………………………………………… 16
8  Structure model building, refinement and validation ……………………………………… 16
9  3D structure of HpSDHs  ………………………………………………………………… 17
9.1  The overall HpSDH structures ………………………………………………………… 17
9.2  Structure of Y210-mutated HpSDHs …………………………………………………… 17
9.3  Structure of Q237-mutated HpSDHs …………………………………………………… 18
9.4  Structure of HpSDH-v2356 …………………………………………………………… 18
10  Results of MtDHQS ……………………………………………………………………… 19
10.1  Purification and crystallization of MtDHQS ………………………………………… 19
10.2  Crystal structure of MtDHQS•NAD complex  ……………………………………… 20
10.3  Crystal structure of MtDHQS•DAHP complex ……………………………………… 20
 Discussion …………………………………………………………………………………… 21
1  HpSDH gene cloning, protein expression and purification  ……………………………… 21
2  Biochemical analysis of HpSDHs ………………………………………………………… 21
3  Crystallization of HpSDH proteins  ……………………………………………………… 21
4  X-ray diffraction data collection and processing  ………………………………………… 22
5  Model building, refinement, and validation ……………………………………………… 22
6  iTC analysis of mutant HpSDHs  ………………………………………………………… 22
7  Structure variation of HpSDH mutants …………………………………………………… 23
8  Structure flexibility of HpSDH-v2356 …………………………………………………… 23
9  Discussion of MtDHQS crystallography  ………………………………………………… 24
9.1  Crystallization of MtDHQS …………………………………………………………… 24
9.2  Domain transition ……………………………………………………………………… 25
9.3  Structure comparison  ………………………………………………………………… 25
10  Conclusion and future direction ………………………………………………………… 26
 Figures and tables …………………………………………………………………………… 27
 Reference …………………………………………………………………………………… 63
Appendix – Crystal Structure of Glucose-1-Phosphate Thymidylyltransferase from Aneurinibacillus thermoaerophilus  …………………………………………………………… 66
Abstract ………………………………………………………………………………………… 68
 Purpose and specific aims ………………………………………………………………… 69
 Material and Methods  …………………………………………………………………… 69
1  Source of AtRmlA protein and ligands  ………………………………………………… 69
2  AtRmlA crystallization screening ………………………………………………………… 69
3  Optimization of crystallization conditions ……………………………………………… 70
4  X-ray diffraction and data processing …………………………………………………… 70
5  AtRmlA model building and refinement ………………………………………………… 70
 Results ……………………………………………………………………………………… 71
1  AtRmlA crystallization …………………………………………………………………… 71
2  X-ray diffraction data collection and processing ………………………………………… 71
3  Structure model building, refinement and validation …………………………………… 72
4  3D structure of AtRmlA  ………………………………………………………………… 72
4.1  Overall structure ……………………………………………………………………… 72
4.2  Structure of active site and ligand binding interactions ……………………………… 72
 Discussion  ………………………………………………………………………………… 73
1  Crystallization of AtRmlA proteins ……………………………………………………… 73
2  X-ray diffraction and model building …………………………………………………… 74
3  Relationship between enzyme mechanism and active site structure  …………………… 74
4  Enzyme engineering for activity enhancement  ………………………………………… 74
 Figures and tables ………………………………………………………………………… 76
 Reference …………………………………………………………………………………… 89

                                

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