臺灣博碩士論文加值系統

Flavin-dependent thymidylate synthase (FDTS) 又稱作ThyX，是一種細菌特有的去氧胸線核苷單磷酸 (dTMP) 合成酵素，主要是將甲基四氫葉酸 (CH2H4folate) 上的甲基轉移至去氧尿苷單磷酸 (dUMP) 含氮鹼基的五號碳上，由於哺乳類動物合成去氧尿苷單磷酸是依靠不同的催化酵素反應，所以ThyX很有可能成為針對細菌進行抑制的標靶目標。在本研究中，主要以解構空腸彎曲桿菌 (Campylobacter jejuni) 的ThyX酵素結構為目的，由於目前沒有相似度高的ThyX作為相位模板，所以我們只能依靠重金屬原子在蛋白質晶體內的特殊吸收光波長來得到相位差，最後是以第17和49號的丙胺酸 (Alanine) 和第110號的白胺酸 (Leucine) 作為蛋胺酸 (Methionine) 點突變的位置，以進行有機態銫 (Selenomethionine) 的晶體培養。利用單一光波長異常散射(Single-wavelength anomalous dispersion, SAD) 的方法，我們成功的解出CjThyX複合物蛋白結構以及突變過後的CjThyX結構。CjhyX結構主要由三個區域所組成，分別是點端螺旋區域、底度螺旋區域和中央α/β混和區域，在中央區域裡發現受質dUMP和FAD與蛋白質結構間的接合。透過與點突變的CjThyX結構以及其他物種的ThyX比較，第86號氨基胍基戊酸 (Arginine) 、第85號羥丁氨酸以及第84號絲氨酸會有結構上的螺旋偏移以進行與受質dUMP的結合。此外，透過活化位的殘基觀察，整個ThyX催化反應是由FAD上的N5氮原子去攻擊dUMP上的C6碳原子所啟動。這些發現對於未來進行以結構為基底的藥物設計方法提供了很多資訊

Flavin-dependent thymidylate synthase (FDTS) ThyX that catalyzes the biosynthesis of TMP by the methylation of uracil moiety of dUMP is crucial for the growth of several important human pathogens including Helicobacter pylori, Mycobacteria tuberculosis, and Campylobacter jejuni. Given its uniqueness, ThyX has been recognized as a promising target for the development of anti-bacterial drug. Here, we determined the Campylobacter jejuni ThyX (CjThyX) structure to 3.0 Å resolution using single-wavelength anomalous dispersion methods on a mutant of CjThyX (A17MA49ML110M). Based on the mutant structure, we are able to solve the wild-type CjThyX．dUMP．FAD as well as A17MA49ML110M．dUMP．FAD to 2.2 Å and 2.45 Å resolution, respectively.
The monomeric wild-type CjThyX structure is composed of 11 α-helices and 4 anti-parallel β-strands. Homotetramer is observed in which four FAD and dUMP molecules are situated at the interface of inter-subunits. In the active site, Ser84, Thr85, and Arg86 clamp the dUMP at a favor position through a helical shift. FAD is bound by the unique ThyX motif near the uridyl ring of dUMP. Structural comparisons with HpThyX and MtThyX indicate that the catalytic sites of ThyX enzyme in different species are highly conserved. The biosynthetic mechanism of CjThyX might also similar to HpThyX which is driven by attacking the C6 position of dUMP via the nitrogen atom N5 of the non-enzymatic nucleophile FADH2.

中文摘要……………………………………………………………….i.
Abstract……………………………………………………………….ii.
Index………………………………………………………………….iii.
List of tables……………………………………………………….v.
List of figures…………………………………………………….vi.
1. Introduction……………………………………………………….1.
2. Materials and Methods
2.1 Cloning and primer design…………………………………….4.
2.2 Cloning and primer design for site-directed mutagenesis study…………………………………………………………………….4.
2.3 Wild-type and A17M-A49M-L110M CjThyX protein expression…………………………………………………………….5.
2.4 SeMet-A17M-A49M-L110M CjThyX protein expression…………………………………………………………….5.
2.5 Protein purification………………………………………….5.
2.6 Gel filtration chromatography……………………………………………………….6.
2.7 Protein condense and dialysis……………………………….6.
2.8 Crystallization and data collection……………………….7.
2.9 Structure determination and refinement………………….8.
2.10 Structure comparisons and analysis……………………….8.
3. Results
3.1 Wild-type CjThyX
3.1.1 Protein expression and purification of wild-type CjThyX………………………………………………………………….9.
3.1.2 Crystallization and data collection of wild-type CjThyX………………………………………………………………….9.
3.2 Site-directed mutagenesis study
3.2.1 Protein expression and purification of A17M-A49M-L110M CjThyX………………………………………………………….10.
3.2.2 Crystallization and data collection of SeMet-A17M-A49M-L110M CjThyX………………………………………………….10.
3.2.3 Crystallization and data collection of Apo and complex form of
A17M-A49M-L110M CjThyX…………………………………………….11.
3.3 CjThyX structure description
3.3.1 Wild-type CjThyX in complex of FAD and dUMP……….12.
3.3.2 FAD and dUMP binding site of wild-type CjThyX structure…………………………………………………………….13.
3.3.3 Apo and complex forms of A17M-A49M-L110M CjThyX….15.
3.4 Comparison of ThyX structures
3.4.1 Conformational differences of wild-type and mutant CjThyX structures………………………………………………….15.
3.4.2 Comparisons of wild-type and mutant CjThyX catalytic site…………………………………………………………………….16.
3.4.3 Structural differences of ThyX in various species………………………………v……………………………….17.
3.4.4 Structural comparisons of ThyX catalytic site with C. jejuni, H. pylori and M.tuberculosis………………………….18.
4. Discussion
4.1 Structural determination of CjThyX via the phase of HpThyX………………………………………………………………….20.
4.2 Site-directed mutagenesis of CjThyX…………………….21.
4.3 Catalytic mechanism of CjThyX…………………………….22.
5. Reference………………………………………………………….24.

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14. Meng, C. Y., Smith, B. L., Bodhidatta, L., Richard, S. A., Vansith, K., Thy, B., Srijan, A., Serichantalergs, O. & Mason, C. J. (2011). Etiology of diarrhea in young children and patterns of antibiotic resistance in Cambodia. Pediatr Infect Dis J 30, 331-5.
15. Zaharevitz, D. W., Anderson, L. W., Malinowski, N. M., Hyman, R., Strong, J. M. & Cysyk, R. L. (1992). Contribution of de-novo and salvage synthesis to the uracil nucleotide pool in mouse tissues and tumors in vivo. Eur J Biochem 210, 293-6.
16. Hunter, J. H., Gujjar, R., Pang, C. K. & Rathod, P. K. (2008). Kinetics and ligand-binding preferences of Mycobacterium tuberculosis thymidylate synthases, ThyA and ThyX. PLoS One 3, e2237.
17. Myllykallio, H., Lipowski, G., Leduc, D., Filee, J., Forterre, P. & Liebl, U. (2002). An alternative flavin-dependent mechanism for thymidylate synthesis. Science 297, 105-7.
18. Leduc, D., Graziani, S., Meslet-Cladiere, L., Sodolescu, A., Liebl, U. & Myllykallio, H. (2004). Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic Bacteria and Archaea. Biochem Soc Trans 32, 231-5.
19. Leduc, D., Escartin, F., Nijhout, H. F., Reed, M. C., Liebl, U., Skouloubris, S. & Myllykallio, H. (2007). Flavin-dependent thymidylate synthase ThyX activity: implications for the folate cycle in bacteria. J Bacteriol 189, 8537-45.
20. Agrawal, N., Lesley, S. A., Kuhn, P. & Kohen, A. (2004). Mechanistic studies of a flavin-dependent thymidylate synthase. Biochemistry 43, 10295-301.
21. Koehn, E. M., Fleischmann, T., Conrad, J. A., Palfey, B. A., Lesley, S. A., Mathews, II & Kohen, A. (2009). An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene. Nature 458, 919-23.
22. Kogler, M., Vanderhoydonck, B., De Jonghe, S., Rozenski, J., Van Belle, K., Herman, J., Louat, T., Parchina, A., Sibley, C., Lescrinier, E. & Herdewijn, P. (2011). Synthesis and Evaluation of 5-Substituted 2'-deoxyuridine Monophosphate Analogues As Inhibitors of Flavin-Dependent Thymidylate Synthase in Mycobacterium tuberculosis. J Med Chem.
23. Esra Onen, F., Boum, Y., Jacquement, C., Spanedda, M. V., Jaber, N., Scherman, D., Myllykallio, H. & Herscovici, J. (2008). Design, synthesis and evaluation of potent thymidylate synthase X inhibitors. Bioorg Med Chem Lett 18, 3628-31.
24. Chernyshev, A., Fleischmann, T. & Kohen, A. (2007). Thymidyl biosynthesis enzymes as antibiotic targets. Appl Microbiol Biotechnol 74, 282-9.
25. Otwinowski, Z., and Minor, W. (1997). Processing of X-ray Diffraction Data Collected in Oscillation Mode, Methods in Enzymology. Macromolecular Crystallography, Pt A 276, 307-326.
26. Adams, P. D., Grosse-Kunstleve, R. W., Hung, L. W., Ioerger, T. R., McCoy, A. J., Moriarty, N. W., Read, R. J., Sacchettini, J. C., Sauter, N. K. & Terwilliger, T. C. (2002). PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 58, 1948-54.strain SS1. Protein Sci.