稻米中3-磷酸甘油醛脫氫酶之蛋白結構與功能分析

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研究生：	田岳衢 Yueh-Chu Tien
論文名稱：	稻米中3-磷酸甘油醛脫氫酶之蛋白結構與功能分析 Structure and Functional Analysis of Glyceraldehyde-3-phosphate Dehydrogenase from Oryza Sativa
指導教授：	張壽麟 Shou-Lin Chang 陳俊榮 Chun-Jung Chen
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：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	65
中文關鍵詞：	3-磷酸甘油醛脫氫酶、醯胺線嘌呤二核苷酸、X光結晶繞射
外文關鍵詞：	GAPDH, G3P, BPG, NAD, NADP, X-ray diffraction, protein structure
相關次數：	點閱：4 下載：0
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稻米細胞質內的3-磷酸甘油醛脫氫酶屬於3-磷酸甘油醛脫氫酶家族的一員，分類編號E.C.1.2.1.12，為醣解作用所需的基本酵素。這個酵素僅使用菸醯胺線嘌呤二核苷酸作為輔酶，對3-磷酸甘油醛進行磷酸化，使其轉變為1,3-二磷酸甘油酸。相較於此，分類編號E.C.1.2.1.13的3-磷酸甘油醛可以使用醯胺線嘌呤二核苷酸或醯胺線嘌呤二核苷酸磷酸作為輔酶，進行逆反應。使用X光結晶繞射法解出稻米細胞質的3-磷酸甘油醛脫氫酶蛋白結構，包括純蛋白結構、與輔酶醯胺線嘌呤二核苷酸結合的蛋白結構以及與硫酸根結合的蛋白結構。與目前其他已被發表的3-磷酸甘油醛脫氫酶結構類似的是，稻米的3-磷酸甘油醛脫氫酶同樣以四個相同次單元聚合物的型態存在，每個次單元也可以分為三個主要區域：催化區域、輔酶結合區域和S-loop區域。此外，輔酶醯胺線嘌呤二核苷酸亦藉由氫鍵與蛋白質結合，一些特定氨基酸形成帶有正電荷的區域，藉此吸引-磷酸甘油醛的磷酸根，在此僅以硫酸根模擬磷酸根的位置。然而，還有一些現象似乎尚未被討論過。第37個氨基酸，苯丙氨酸，在醯胺線嘌呤二核苷酸上方形成一個瓶頸，或許可以增進醯胺線嘌呤二核苷酸的結合能力，然而這個假設還須進一步的證實。除此之外，雖然脯氨酸193與天冬氨酸35已在先前的研究成果中，被證實是造成對醯胺線嘌呤二核苷酸具有專一性的關鍵氨基酸，但從結構上分析，苯丙氨酸37也對醯胺線嘌呤二核苷酸的專一性具有關鍵作用。藉由安基酸序列的比對，可以發現脯氨酸與天冬氨酸存在於所有對醯胺線嘌呤二核苷酸具有專一性的蛋白質中，但苯丙氨酸37僅存在於較高等生物的3-磷酸甘油醛脫氫酶。這或許意味著某種演化上的結果，並可能再不同物種間造成不同的生理現象。

Abstract.......................................................................................................................ii
Table of Contents........................................................................................................iv
List of Figures.............................................................................................................v
List of Tables..............................................................................................................vi

Chapter 1. Introduction...............................................................................................1
1.     Glycolysis pathway.....................................................................................1
2.     Introduction of GAPDH..............................................................................1
3.     Catalytic mechanism...................................................................................2
4.     Subfamily of GAPDH.................................................................................3
5.     Characterization of cytoplasm GAPDH......................................................4
6.     Issues of this project....................................................................................4

Chapter 2. Materials and Methods..............................................................................5
 Clone GAPDH from Oryza Sativa..............................................................5
 Expression and purification of GAPDH.....................................................6
 Crystallization of GAPDH...........................................................................7
 X-ray Data collection and process...............................................................8
 Structure determination and refinement.......................................................8

Chapter 3. Results......................................................................................................10
3.1     Sequence and expression of GAPDH from Oryza sativa...........................10
3.2     Crytal, structure determination and model quality.....................................11
3.3     Native structure description........................................................................12
3.4     NAD-binding region...................................................................................14
3.5     Comparison between NAD-free and NAD-bound structure.......................15
3.6     Ps and Pi sites of OsGAPDH .....................................................................15
3.7     Comparison between OsGAPDH and SoGAPDH......................................16

Chapter 4. Discussion.................................................................................................19
4.1     NAD-free structure and NAD-binding structure.........................................19
4.2     Compare NAD-binding and NADP-binding region....................................20
4.3     Sulfate binding region.................................................................................22

Chapter 5. Conclusion................................................................................................23

Reference....................................................................................................................25

Figures and Tables......................................................................................................28

                                

Andrade, J., Pearce, S. T., Zhao, H., Barros, M., 2004. Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules. Biochem. J. 384,327–336.
Baalmann, E., Scheibe, R., Cerf, R., Martin, W., 1996. C-terminal truncation of spinach chloroplast NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase prevents inactivation and reaggregation. Plant Mol Biol. 32, 505-513.
BAIBAI, T., OUKHATTAR, L., MOUTAOUAKKIL A.., SOUKRI, A., 2007. Purification and Characterization of Glyceraldehyde-3-phosphate Dehydrogenase from European Pilchard Sardina pilchardus. Acta Biochimica et Biophysica Sinica. 39(12), 947–954.
Buehner, M., Ford, G. C., Moras, D., Olsen, K. W., Rossmann, M. G., 1975. Studies of asymmetry in the three-dimensional structure of lobster D-glyceraldehyde-3-phosphate dehydrogenase. J. Biol. Chem. 250(23), 9137-9162.
Cerff, R., Chambers, S., 1979. Subunit structure of higher plant glyceraldehyde-3-phosphate dehydrogenase. J. Biol. Chem. 254, 6094-6098.
Clermont, S., Corbier, C., Mely, Y., Gerard, D., Wonacott, A., Branlant G.,1993. Determinants of Coenzyme Specificity in Glyceraldehyde-3-phosphate Dehydrogenase: Role of the Acidic Residue in the Fingerprint Region of the Nucleotide Binding Fold. Biochemistry. 32, 10178-10184.
Didierjean, C., Corbier, C., Fatih, M., Favier, F., Boschi-Muller, S., Branlant, G., Aubry, A., 2003. Crystal Structure of Two Ternary Complexes of Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from Bacillus stearothermophilus with NAD and D-Glyceraldehyde 3-Phosphate. The Journal of Biological Chemistry. 278(15), 12968–12976.
Didierjean, C., Rahuel-Clermont, S., Vitoux, B., Dideberg, O., Branlant G., Aubry, A., 1997. A Crystallographic Comparison Between Mutated Glyceraldehyde-3-phosphate Dehydrogenases from Bacillus stearothermophilus Complexed with Either NAD. or NADP. J. Mol. Biol. 268, 739-759.
Engel, M., Seifert, M., Theisinger, B., Seyfert, U., Welter, C., 1998. Glyceraldehyde-3-phosphate dehydrogenase and Nm23-H1/nucleoside diphosphate kinase A. J. Biol. Chem. 273, 20058–20065.
Fermani, S., Ripamonti, A., Sabatino, P., Zanotti, G., Scagliarini, S., Sparla, F., Trost, P., Pupillo, P., 2001. Crystal Structure of the Non-regulatory A4 Isoform of Spinach Chloroplast Glyceraldehyde-3-phosphate Dehydrogenase Complexed with NADP. J. Mol. Biol. 314, 527-542.
Fermani, S., Sparla, F., Falini, G., Martelli, P.L., Casadio, R., Pupillo, P., Ripamonti, A., Trost, P., 2007. Molecular mechanism of thioredoxin regulation in photosynthetic A2B2-glyceraldehyde-3-phosphate dehydrogenase. PNAS. 104(26), 11109–11114.
Harrigan, P. J., Trenthan, D. R., 1974. Kinetic studies on oxidized nicotinamide-adenine dinucleotide-facilitated reactions of D-Glyceraldehyde-3-phosphate dehydrogenase. Biochem. J. 143, 353–363.
Kim, H., Feil, I.K., Verlinde, C.L., Petra, P.H., Hol, W.G., 1995. Crystal structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Leishmania mexicana: implications for structure-based drug design and a new position for the inorganic phosphate binding site. Biochemistry .34(46), 14975-14986.
Kim, J. W., Dang, C. V., 2005. Multifaceted roles of glycolytic enzymes. Trends Biochem. Sci. 30, 142–150.
Liu, L., Huskey, W.P., 1992. Progress in Establishing the Rate-Limiting Features and Kinetic Mechanism of the Glyceraldehyde-3-phosphate Dehydrogenase Reaction. Biochemistry. 31, 6898-6903.
Meunier, J.-C., Dalziel, K., 1978. Kinetic Studies of Glyceraldehyde-3-Phosphate Dehydrogenase from Rabbit Muscle. J. Biochem. 82, 483-492.
Nakagawa, T., Hirano, Y., Inomata, A., Yokota, S., Miyachi, K., Kaneda, M.,Umeda, M., Furukawa, K., Omata, S., and Horigome, T., 2003. Participation of a fusogenic protein, glyceraldehyde-3-phosphate dehydrogenase, in nuclear membrane assembly. J. Biol.Chem. 278, 20395–20404.
Pupillo, P., Faggiani, R., 1979. Subunit structure of hree glyceraldehyde-3-phosphate dehydrogenases of some flowering plants. Arch. Biochem. Biophys. 154, 475-482.
Pupillo P., Giuliani P. G., 1975. The reversible depolymerization of spinach chloroplast glyceraldehyde-phosphate dehydrogenease. Interaction with nucleotides and dithiothreitol. Eur. J. Biochem. 51, 475–482.
Scagliarini, S., Trost, P., Pupillo, P., 1998. The non-regulatory isoform of NADP(H)-glyceraldehyde-3-phosphate dehydrogenase from spinach chloroplasts. J. Exp. Bot. 49, 1307-1315.
Skarzynski, T., Moody, P. C. E., Wonacott, J. A., 1987. Structure of holo-glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus at 1.8A resolution. J. Mol. Biol.. 193, 171–187.
Sparla F., Pupillo P., Trost P., 2002. The C-terminal extension of glyceraldehyde-3-phosphate dehydrogenase subunit B acts as an autoinhibitory domain regulated by thioredoxins and nicotinamide adenine dinucleotide. J. Biol. Chem. 277, 44946–44952.
Tanner, J. J., Hecht, M. R., Krause L. K., 1996. Determinants of Enzyme Thermostability Observed in the Molecular Structure of Thermus aquaticus D-Glyceraldehyde-3-phosphate Dehydrogenase at 2.5 Å Resolution. Biochemistry. 35 (8), 2597 -2609.
Tisdale, E. J., 2001. Glyceraldehyde-3-phosphate is required for vesicular transport in the early secretory pathway. J. Biol. Chem. 276, 2480–2486.
Tisdale, E. J., Kelly, C., Artalejo, C. R., 2004. Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity. J. Biol. Chem. 279, 54046–54052.
Yun, M., Park, C.-G., Kim, J.-Y., Park, H.-W., 2000. Structural Analysis of Glyceraldehyde 3-Phosphate Dehydrogenase from Escherichia coli: Direct Evidence of Substrate Binding and Cofactor-Induced Conformational Changes. Biochemistry. 39, 10702-10710.
Zheng, L., Roeder, R. G., Luo, Y., 2003. S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component. Cell. 114, 255–266.

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