Computational Study of the Catalytic Mechanism of Ketol–Acid Reductoisomerase of Sso-ilvC2 Protein

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：64

、訪客IP：18.116.86.134

姓名

翁聖崴(Sheng-Uei Weng) 查詢紙本館藏

畢業系所

化學學系

論文名稱

(Computational Study of the Catalytic Mechanism of Ketol–Acid Reductoisomerase of Sso-ilvC2 Protein)

相關論文

★ 嗜甲烷菌內甲烷單氧化酵素中催化反應中心三核銅模擬分子之合成與光譜分析	★ 烷烴氧化菌及氧化酵素之純化與功能性探討
★ 以電腦模擬研究香蕉型液晶元的分子交互作用力	★ 利用時間相關的電子密度泛函理論研究反式-二苯乙烯胺的光化學行為
★ 以生物資訊法研究穩定Asparagine在左手螺旋形下的交互作用力	★ 葛蘭氏陰性菌脂質A之結構研究
★ 五苯荑衍生之苯乙炔寡聚物之合成與光物理性質研究	★ 紫質三元件系統的金屬化作用對遠端氫鍵調控的影響
★ 非鍵結作用力的理論研究： (1)質子化與氧化三元件系統遠端調控氫鍵的比較 (2)π- π與CH- π作用力的取代基效應	★ 利用時間相關的密度泛涵理論研究HBI分子及其衍生物在第一激發態的位能曲線
★ Replica-Exchange分子動態模擬法研究類澱粉胜肽25-35 嵌入膜與折疊的行為	★ 抗菌胜肽資料庫分析及利用分子動態模擬法探討抗菌胜肽Indolicidin於生物膜上的行為
★ 網頁圖形界面在分子模擬上的應用	★ 類澱粉胜肽Abeta(25-35) 序列影響該類胜肽在水-膜環境下的組態: 強調多樣性的神經毒性
★ 以分子動態模擬法研究陽離子-負電磷脂質雙層的配位網絡結構：延伸應用於膜融合機制	★ 染料敏化太陽能電池吸光性質的計算研究

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

摘要(中)

支鏈型胺基酸合成路徑 (branched chain amino acids biosynthetic pathway) 涉及了三種不同的酵素來參與整個化學反應。酮酸還原異構? (ketol-acid reductoisomerase) 是支鏈型胺基酸合成路徑中參與反應的第二個酵素。這個酵素對於開發新型的除草劑與抗生素是一個很好的目標，因為它不存在於人體與動物之中。酮酸還原異構?的催化總反應依序為，第一步，與鎂離子有關的烷基轉移；第二步，利用菸鹼醯胺腺嘌呤二核?酸磷酸或菸鹼醯胺腺嘌呤二核?酸NAD(P)H 進行酮基還原。然而，對於酮酸還原異構?的反應機制依然是不夠清楚的，而且對於鎂離子在酵素中扮演的角色與總反應的起始劑為何都是不清楚的。在本篇研究中，我們的研究對象為Sso-ilvC2 蛋白，它是硫化葉菌 (Sulfolobus solfataricus) 的酮酸還原異構?，我們利用分子動態柔性擬合 (molecular dynamic flexible fitting) 技術與低溫電子顯微鏡 (cryo-EM) 影像 (解析度 = 3.4 A) 來建立Sso-ilvC2 蛋白的三維結構。我們利用密度泛函理論的計算方法來探討，在不同情況下的反應物，對於甲基轉移所需的活化能。在我們的研究中發現，反應物上的酮基先被質子化後，在進行甲基轉移，所需要的活化能是最低的 (21.5 kcal/mol)。

摘要(英)

The ketol-acid reductoisomerase (KARI) is an enzyme necessary for the branched chain amino acids (BCAA) biosynthetic pathway. KARIs catalyze an alkyl-migration of substrate followed by a ketol-acid reduction in terms of NAD(P)H. However, the KARI reaction mechanism is remained unclear; in particular, the roles of Mg2+ ion pair in the catalysis and how the KARI reaction is initialized are still unknown. In this study, we obtain the 3D structure of Sso-ilvC2 protein, a KARI from Sulfolobus solfataricus, from the cryo-EM (resolution = 3.4 A) in terms of the molecular dynamic flexible fitting (MDFF) technique. We employ the first principle DFT calculations to investigate the activation energy of methyl migration under different catalysis conditions of substrate based on the active site structure of Sso-ilvC2 protein obtained from the cryo-EM structure refined by MD simulations. We observed that the substrate when its ketone group is protonated has lowest active energy (21.5 kcal/mol) for the methyl migration.

關鍵字(中)

★ 酮酸還原異構?
★ 硫化葉菌
★ 低溫電子顯微鏡影像

關鍵字(英)

★ Ketol–Acid Reductoisomerase
★ Sso-ilvC2
★ cryo-EM

論文目次

摘要 i
Abstract ii
致謝 iii
Contents iv
List of Figures v
Chapter 1: Introduction 1
Chapter 2: Computational Methods 5
Chapter 3: Results and Discussion 12
3-1 Generated the testing protein model 12
3-2 Performance of MDFF 15
3-3 Performance of Rosetta 18
3-4 Performance of Iterative MDFF-Rosetta 18
3-5 3D Structure of Sso-ilvC2 obtained from the cryo-EM 21
3-6 MD simulations of Sso-ilvC2 Complex Structure obtained from the cryo-EM 25
3-7 First-Principle Investigation of the Catalytic Mechanism of Ketol–Acid Reductoisomerase (KARI) 27
Chapter 4: Conclusions 40
References 41

參考文獻

1. Garcia, M. D.; Nouwens, A.; Lonhienne, T. G.; Guddat, L. W., Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families. Proceedings of the National Academy of Sciences 2017, 114 (7), E1091-E1100.
2. Arfin, S. M.; Umbarger, H. E., Purification and Properties of the Acetohydroxy Acid Isomeroreductase of Salmonella typhimurium. Journal of Biological Chemistry 1969, 244 (5), 1118-1127.
3. Chunduru, S. K.; Mrachko, G. T.; Calvo, K. C., Mechanism of ketol acid reductoisomerase. Steady-state analysis and metal ion requirement. Biochemistry 1989, 28 (2), 486-493.
4. Thomazeau, K.; Dumas, R.; Halgand, F.; Forest, E.; Douce, R.; Biou, V., Structure of spinach acetohydroxyacid isomeroreductase complexed with its reaction product dihydroxymethylvalerate, manganese and (phospho)-ADP-ribose. Acta Crystallographica Section D 2000, 56 (4), 389-397.
5. Proust-De Martin, F.; Dumas, R.; Field, M. J., A Hybrid-Potential Free-Energy Study of the Isomerization Step of the Acetohydroxy Acid Isomeroreductase Reaction. Journal of the American Chemical Society 2000, 122 (32), 7688-7697.
6. Mrachko, G. T.; Chunduru, S. K.; Calvo, K. C., The pH dependence of the kinetic parameters of ketol acid reductoisomerase indicates a proton shuttle mechanism for alkyl migration. Archives of Biochemistry and Biophysics 1992, 294 (2), 446-453.
7. Sonya, T.; M., P. M.; Volker, S.; A., L. J.; W., G. L.; Gerhard, S., Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol–Acid Reductoisomerase. Chemistry – A European Journal 2016, 22 (22), 7427-7436.
8. M., P. K.; David, T.; Shan, Z.; Ajit, K.; Mario, G.; You, L.; A., S. M.; P., M. R.; Gerhard, S.; W., G. L., Crystal Structures of Staphylococcus aureus Ketol?Acid Reductoisomerase in Complex with Two Transition State Analogues that Have Biocidal Activity. Chemistry – A European Journal 2017, 23 (72), 18289-18295.
9. Bartesaghi, A.; Merk, A.; Banerjee, S.; Matthies, D.; Wu, X.; Milne, J. L. S.; Subramaniam, S., 2.2 A resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor. Science 2015, 348 (6239), 1147-1151.
10. Merk, A.; Bartesaghi, A.; Banerjee, S.; Falconieri, V.; Rao, P.; Davis, M. I.; Pragani, R.; Boxer, M. B.; Earl, L. A.; Milne, J. L. S.; Subramaniam, S., Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery. Cell 2016, 165 (7), 1698-1707.
11. Best, R. B.; Zhu, X.; Shim, J.; Lopes, P. E. M.; Mittal, J.; Feig, M.; MacKerell, A. D., Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ(1) and χ(2) dihedral angles. Journal of chemical theory and computation 2012, 8 (9), 3257-3273.
12. Phillips James, C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel Robert, D.; Kale, L.; Schulten, K., Scalable molecular dynamics with NAMD. Journal of Computational Chemistry 2005, 26 (16), 1781-1802.
13. Humphrey, W.; Dalke, A.; Schulten, K., VMD: Visual molecular dynamics. Journal of Molecular Graphics 1996, 14 (1), 33-38.
14. Bender, B. J.; Cisneros, A.; Duran, A. M.; Finn, J. A.; Fu, D.; Lokits, A. D.; Mueller, B. K.; Sangha, A. K.; Sauer, M. F.; Sevy, A. M.; Sliwoski, G.; Sheehan, J. H.; DiMaio, F.; Meiler, J.; Moretti, R., Protocols for Molecular Modeling with Rosetta3 and RosettaScripts. Biochemistry 2016, 55 (34), 4748-4763.
15. Lindert, S.; Alexander, N.; Wotzel, N.; Karaka?, M.; Stewart, P. L.; Meiler, J., EM-Fold: De novo atomic-detail protein structure determination from medium resolution density maps. Structure(London, England:1993) 2012, 20 (3), 464-478.
16. DiMaio, F.; Song, Y.; Li, X.; Brunner, M. J.; Xu, C.; Conticello, V.; Egelman, E.; Marlovits, T. C.; Cheng, Y.; Baker, D., Atomic-accuracy models from 4.5-A cryo-electron microscopy data with density-guided iterative local refinement. Nature Methods 2015, 12, 361.
17. DiMaio, F.; Tyka, M. D.; Baker, M. L.; Chiu, W.; Baker, D., Refinement of Protein Structures into Low-Resolution Density Maps using Rosetta. Journal of molecular biology 2009, 392 (1), 181-190.
18. Lindert, S.; McCammon, J. A., Improved cryoEM-Guided Iterative Molecular Dynamics–Rosetta Protein Structure Refinement Protocol for High Precision Protein Structure Prediction. Journal of Chemical Theory and Computation 2015, 11 (3), 1337-1346.
19. Trabuco, L. G.; Villa, E.; Schreiner, E.; Harrison, C. B.; Schulten, K., Molecular Dynamics Flexible Fitting: A practical guide to combine cryo-electron microscopy and X-ray crystallography. Methods (San Diego, Calif.) 2009, 49 (2), 174-180.
20. C., A. H., Constant pressure molecular dynamics simulation: The Langevin piston method. The Journal of Chemical Physics 1995, 103 (11), 4613-4621.
21. Ryckaert, J.-P.; Ciccotti, G.; Berendsen, H. J. C., Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. Journal of Computational Physics 1977, 23 (3), 327-341.
22. Becke, A. D., Density?functional thermochemistry. III. The role of exact exchange. The Journal of Chemical Physics 1993, 98 (7), 5648-5652.
23. Lee, C.; Yang, W.; Parr, R. G., Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Physical Review B 1988, 37 (2), 785-789.
24. Petersson, G. A.; Al-Laham, M. A., A Complete Basis Set Model Chemistry. II. Open-Shell Systems and the Total Energies of the First-Row Atoms. J. Chem. Phys. 1991, 94, 6081.
25. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A. V.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J. Gaussian 16 Rev. B.01, Wallingford, CT, 2016.
26. J., T.; M., P., Continuous surface charge polarizable continuum models of solvation. I. General formalism. The Journal of Chemical Physics 2010, 132 (11), 114110.
27. Trabuco, L. G.; Villa, E.; Mitra, K.; Frank, J.; Schulten, K., Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics. Structure (London, England : 1993) 2008, 16 (5), 673-683.
28. Singharoy, A.; Teo, I.; McGreevy, R.; Stone, J. E.; Zhao, J.; Schulten, K., Molecular dynamics-based refinement and validation for sub-5 A cryo-electron microscopy maps. eLife 2016, 5, e16105.
29. Vanommeslaeghe, K.; D Mackerell, A., Automation of the CHARMM general force field (CGenFF) I: Bond perception and atom typing. 2012; Vol. 52.
30. Vanommeslaeghe, K.; Raman, P.; D Mackerell, A., Automation of the CHARMM General Force Field (CGenFF) II: Assignment of bonded parameters and partial atomic charges. 2012; Vol. 52.
31. Chan, K.-Y.; Gumbart, J.; McGreevy, R.; Watermeyer, Jean M.; Sewell, B. T.; Schulten, K., Symmetry-Restrained Flexible Fitting for Symmetric EM Maps. Structure 2011, 19 (9), 1211-1218.
32. Brinkmann-Chen, S.; Flock, T.; Cahn, J. K.; Snow, C. D.; Brustad, E. M.; McIntosh, J. A.; Meinhold, P.; Zhang, L.; Arnold, F. H., General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH. Proceedings of the National Academy of Sciences of the United States of America 2013, 110 (27), 10946-51.
33. Cahn, J. K.; Brinkmann-Chen, S.; Spatzal, T.; Wiig, J. A.; Buller, A. R.; Einsle, O.; Hu, Y.; Ribbe, M. W.; Arnold, F. H., Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases. The Biochemical journal 2015, 468 (3), 475-84.

指導教授

蔡惠旭(Hui-Hsu Tsai)

審核日期

2018-8-14

推文