稻米α-澱粉酶/枯草桿菌蛋白酶抑制素之X光晶體結構與功能研究

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研究生：	彭文彥 Wen-Yan Peng
論文名稱：	稻米α-澱粉酶/枯草桿菌蛋白酶抑制素之X光晶體結構與功能研究 Structure and function study of rice bifunctional α-amylase/subtilisin inhibitor from Oryza sativa
指導教授：	吳文桂 Wen-Guey Wu 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：	2007
畢業學年度：	95
語文別：	英文
論文頁數：	65
中文關鍵詞：	稻米、澱粉酶抑制素、枯草桿菌酶抑制素
外文關鍵詞：	rice, amylase inhibitor, subtilisin inhibitor
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稻米種子富有許多蛋白酶的抑制素，其中有些對兩種不同蛋白酶具有抑制功能的抑制素，例如alpha-澱粉酶/枯草桿菌酶抑制素、胰蛋白酶/胰凝乳蛋白酶抑制素等。這些抑制素通常在生長中的種子或其他器官裡被表現出來，並且在植物中扮演了防禦系統上的重要角色。更進一步的，不只在植物生長中扮演了重要的角色，還廣泛地提供了對病蟲害的抵禦作用。然而，雖然一些抑制素在其他物種已經有了高解析度的蛋白結構或者對目標蛋白的複合物結構，稻米卻還尚未有相關的蛋白結構被發表出來。所以在這項研究當中，我們從米糠中純化出了純質的稻米alpha-澱粉酶/枯草桿菌酶抑制素(RASI, 18.9kDa)，並利用懸式蒸發擴散法培養出適合的晶體。根據X光在解析度1.80Å繞射數據，所得的晶體晶格屬於P21212空間群，三軸長為a=79.99Å, b=62.95Å 和 c=66.70Å。其初步分析指出在一單位晶格中有兩個蛋白分子，並含有44.05百分比的溶劑。每一蛋白分子具有五對反式，共十個beta-摺板結構。RASI在植物種子中的詳細作用目前還不清楚。由於能夠抑制枯草桿菌酶與某些細菌的alpha-澱粉酶，故被認為扮演防禦機制的角色。在此研究中，更發現可能與硫氧還原蛋白有作用，但目前尚未有文獻支持其直接的相關性。其結構與其他物種比起來，的確有明顯的不同，一如其序列、酵素活性、物化性質和生理性質上也有明顯的區別。這裡我們呈現一個成功純化與成晶的RASI蛋白，目的在於提供全世界最豐富的三種糧食作物之一的稻米，他的基本結構資訊與一些抑制作用的功能討論。我們可以比較RASI與其他相同性質的蛋白，以得到更多有關蛋白功能的資訊。

Rice seeds are rich sources of different proteinaceous inhibitors with characteristic bifunctional enzyme-inhibiting activities such as α–amylase/subtilisin and trypsin/chymotrypsin inhibitors. These inhibitors were generally expressed in developing seeds and other organs, and were thought to play an important role in plant’s defense system. It was generally believed that these inhibitors not only playing a role in plant development but also conferring a broad spectrum of resistance against pests and pathogens. Nevertheless, high-resolution structures of these inhibitors and in complex with target enzymes are available in other species but not in rice. Therefore, in this study, we purified　RASI (rice alpha-amylase/subtilisin inhibitor, 18.9 kDa) from rice bran into homogenous and crystallized using the handing　drop vapor　diffusion method. According to X-ray diffraction of RASI crystals at resolution 1.80 Å, the unit cell belongs to space group P21212 with parameters a=79.99Å, b=62.95Å and c=66.70Å. Preliminary analysis of the protein indicate that there are two RASI molecules in an asymmetric unit with a solvent content of 44.05 %. One monomer contains 10 beta-sheet structures which are five anti-beta-sheet pairs. Detail of the function of RASI in plant seeds seems to remain unclear. Because of its inhibition to Subtilisin and alpha-amylase of certain bacteria, RASI was considered playing a defense role. In this research, we find that RASI might have some interaction to thioredoxin. But there were still no reference which improved the relationship directly. Despite it close structural relationship to other cereals showed distinct differences in sequences, in enzymatic and physico-chemical properties as well as the physiological expression to other species. Here we describe the successful purification and crystallographic characterization of RASI, and the goal of this project is to provide an alternative structural bases and ultimately functional illustration of enzyme-inhibitor interactions in rice plant which is one of the three most valuable cereal crops grown worldwide. In comparing between RASI and the homologous proteins, we can get more information about the protein functions.

ABSTRACT (Chinese)..................................................i
ABSTRACT..........................................................iii
TABLE OF CONTENTS..................................................vi
Chapter 1 INTRODUCTION..............................................1
1.1 alpha-Amylase and Subtilisin.....................................1
1.1.1 Introduction to alpha-Amylase................................1
1.1.2 Introduction to Subtilisin...................................2
1.2 Amylase Inhibitors...............................................2
1.2.1 Introduction to alpha-Amylase Inhibitor.......................2
1.2.2 Classification of Bifunctional Amylase Inhibitor..............3
1.3 Kunitz Soybean Trypsin Inhibitor Families.........................4
1.3.1 Kunitz Soybean Trypsin Inhibitor (KTI)........................4
1.3.2 Barley α-Amylase/Subtilisin Inhibitor (BASI)..................5
1.3.3 Rice α-Amylase/Subtilisin Inhibitor (RASI)....................6
Chapter2 MATERIALS AND METHODS.......................................8
2.1 Materials.......................................................8
2.1.1 Medicine and Kits...........................................8
2.1.2 Instrument..................................................8
2.2 Protein Purification and Identification...........................9
2.2.1 RASI Large-scale Expression and Purification..................9
2.2.2 High Performance Liquid Chromatography (HPLC).................9
2.3 Crystallization................................................10
2.3.1 Variables in Protein Crystallization Experiment..............10
2.3.2 The Theorem of Crystallization...............................11
2.3.3 Vapor Diffusion Method......................................12
2.3.4 Crystal....................................................12
2.4 X-ray Data Collection and Processing.............................12
2.5 Phasing........................................................13
2.5.1 Molecular Replacement (MR)..................................13
2.5.2 Anomalous Scattering Method.................................14
2.5.3 Isomorphous Replacement Method..............................15
2.6 Model Building and Refinement...................................16
2.6.1 Solvent Content.............................................16
2.6.2 Model Building..............................................16
2.6.3 Refinement by using CNS......................................23
2.7 Activity Assay..................................................25
2.7.1 Subtilisin and RASI Assay....................................25
2.7.2 α-Amylase and RASI Assays...................................26
Chapter3 RESULTS AND DISCUSSION....................................27
3.1 Results........................................................27
3.1.1 RASI Protein and Crystal....................................27
3.1.2 Data Collection.............................................27
3.1.3 Model Building and Refining.................................28
3.2 RASI Characteristics............................................28
3.2.1 Sequence alignment with Other Species........................29
3.2.2 RASI Dimer Structure.......................................30
3.2.3 RASI Monomer Structure......................................31
3.2.4 Calcium Ion of Chain A.......................................31
3.2.5 Ramachandran Plot and B-factor Plot..........................31
3.3 Structure Compare of RASI and Other Species.....................32
3.3.1 Structure Comparison with BASI...............................32
3.3.2 Disordered Loops in RASI.....................................33
3.4 Disulfide Bonds.................................................34
3.4.1 Compare with BASI...........................................35
3.5 Amylase Binding Interface Residues...............................35
3.5.1 Comparison with 1AVA, 2IWT and RASI...........................36
3.5.2 Comparison with Q162 (RASI), E168 (1AVA-D) and E168 (2IWT-B)...36
Chapter4 DISCUSSION................................................38
4.1 Disulfide C140-C144.............................................38
4.2 Disordered Loops................................................39
4.3 RASI Functions..................................................40
REFERENCES.........................................................41

List of Tables
Table1 Crystal Diffraction Statistics of RASI........................45
Table2 RASI and BASI RMSD............................................46

List of Illustrations
Figure1 SDS-PAGE...................................................48
Figure2 Phase Diagram...............................................49
Figure3 RASI Crystal................................................49
Figure4 Visual Inspection of the Best Solution.......................50
Figure5 Inhibitory Action of RASI on Subtilisin......................50
Figure6 3D-Structure Plot of RASI Monomer Structure..................51
Figure7 Sequence Alignment of RASI and Other Species..................52
Figure8a 3D-Structure Plot of RASI Dimer Structure..................53
Figure8b Vertical View of RASI Dimer Structure......................53
Figure9 RASI Dimer Interface View...................................54
Figure10 Positions of Disulfide Bond and Active Site in RASI Structure..55
Figure11 Stereo Picture of Superimposition Structure with RASI and BASI (1AVA-D)..................................................56
Figure12 Electrostatics Plot of RASI Dimer Structure...............57
Figure13 Ramachandran Plot of RASI Structure.........................58
Figure14 B-factor Plot of RASI Structure.............................59
Figure15 Stereo Plot of Superimposition Structure....................60
Figure16 Superimposition Plot of the Disordered Loops.................61
Figure17a Electron Density Map of RASI Disulfide C41-C90............62
Figure17b Electron Density Map of RASI Disulfide C41-C90............62
Figure18a C140-C144 of RASI (oxidation form) Compare with C144-C148 of BASI......................................................63
Figure18b C140-C144 of RASI (reduction form) Compare with C144-C148 of BASI......................................................63
Figure19a RASI, 2IWT-B, 1AVA-D and 1AVA-B Superimposition Plot.....64
Figure19b Partial View of Superimposition Plot.......................64
Figure20 Superimposition Plot of Q162 of RASI, E168 of 1AVA-D and E168 of 2IWT-B.................................................65

                                

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