以X光晶體結構方法來佐證醣基轉換反應：Trichoderma harzianum ETS323木聚糖[]與木聚寡糖複合物研究

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研究生：	劉恩鴻 En-Hung Liu
論文名稱：	以X光晶體結構方法來佐證醣基轉換反應：Trichoderma harzianum ETS323木聚糖[]與木聚寡糖複合物研究 Complex structure of Xylanase with xylo-oligosaccharides from Trichoderma harzianum ETS323 : Structural evidence for glycosyl transferase reaction
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：	50
中文關鍵詞：	木聚糖[] 、反應物結合、木聚寡糖、複合物結構、糖?合成?
外文關鍵詞：	Xylanase, substrate binding, xylooligosaccharide, complex structure, glycosynthase
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以X光晶體結構方法從屬於家族11糖基水解?的木聚糖?與木聚寡糖複合物高解析度三維分子結構中得到關於木聚寡糖與酵素結合的重要資訊。由兩個木聚三糖得來的木聚六糖電子密度中顯示木聚糖?具有糖基轉機?的活性。這是第一次從蛋白質結構中看到反應物停留在家族11糖基水解?的全部六個反應單元中，發現幾個重要殘基扮演非常重要的角色。Arg122產生了方向的變異，提供了固定反應物的氫鍵，如果Arg122不產生方向上的轉變是無法提供在反應位 (-1)的氫鍵固定功能。其他接近反應區的殘基也適當提供氫鍵增加木聚寡糖反應物在反應區域的結合穩定性，而W18、Y179與Y96則利用本身具有的苯環與木糖環在反應位 (-2,+2和+3)的位置形成芳香性π軌域重疊反應，辨認適當種類的反應物進行結合。進行糖基轉換的兩個重要殘基分別是Glu86與Glu177，前者作為親核性(鹼性)端，後者則是質子提供(酸性)端，構成了連接兩個木聚寡糖間的配糖鍵。其它木聚糖?與木聚寡糖複合物的結構成果也都符合先前研究工作的發現。綜合以上的結構證據，可以解釋在酵素動力學上所得到的結果，並提供一些方向做為改良酵素活性、提高對熱忍受與設計糖?合成?的參考

The high resolution (1.2-1.4 ?) complex three-dimensional structures of xylanase (family 11) provide the xylooligosaccharide substrates binding information by X-ray crystallography. The electron density of xylohexaose from two xylotriose show GH-11 xylanase have glycosyltransferase activity in the xylotriose soaking data. It is the first time to capture that the 6 subsites abound with substrates. Arg122 changed the orientation to offer a hydrogen bond to fix xylooligosaccharides in the catalytic site. Other residues were close to the catalytic pocket also form hydrogen bonds to stabilize the substrates. W18 , Y179 and Y96 interact with the xylopyranose rings by aromatic stacking at the subsite (-2,+2 and +3) to recognize substrate binding actions. Two glutamic acids, Glu86 and Glu177 are a nucleophile (base) and a proton donor (acid), interact with substrates at subsite (-1) to build up a glycosidic bond to connect two xylotriose sugars. Other complex structure datas confirm previous reports. The structural information can explain the GH-11 xylanase kinetic data and offer a direction to improve the specify activity, heat tolerant and glycosynthase activity.

Table of Contents
封面    i
摘要    ii
Abstract    iii
致謝    iv
Table of Contents    v
List of Figures    vi
List of Tables    vii

 Chapter. 1 Introduction    1
 1.1 Xylanase-Function    1
 1.2 Xylanase-Family    1
 1.3 GH-11 and GH-10 xylanase    2
 1.4 Xylanases Applications    3
 1.5 The mechanisms of glycosyl hydrolases     3
 1.6 Sugar-Binding Subsites in Glycosyl Hydrolases    4
 1.7 GH-11 Xylanase Structures    4

 Chapter. 2 Materials and Methods    7
 2.1 Protein Purification    7
 2.2 Crystallization Screening    8
 2.3 Xylooligosaccharide Preparation    9
 2.4 Substrate Soaking    9
 2.5 X-ray Data Collection and Processing    10
 2.6 Structure Determination and Refinement    11
 2.7 TLC-End Product Analysis    13

 Chapter. 3 Results and Discussion    14
 3.1 Xylanase crystallization    14
 3.2 Crystallographic Data    14
 3.3 Xylanase Native Structure    15
 3.4 Structures of different substrates and time scale    15
 3.5 The xylohexaose electron density of the xylotriose complex    17
 3.6 Substrates specificity    18
 3.7 End Product Analysis    20
 3.8 The Substrate Binding of glycosynthase     21

 Chapter. 4 Conclusion    23

 Bibliography    25
 Figures and Tables    28
List of Figures
Figure 1: Structure of xylan and the sites of its attack by xylanolytic enzymes.[1]    28
Figure 2: Structures of xylo-oligosaccharides.    28
Figure 3: Structures from different family xylanase.    29
Figure 4: Xylanase proteins collected from the final purification step was analyzed by 12.5 % SDS-PAGE.  Lane 1, molecular-weight markers in kDa; lane 2, 10 ug xylanase, 4X sample buffer, boiled at 100 °C for 15 minutes.  Protein bands were revealed by Coomassie Blue R-250. Low range molecular weight standards were used as size markers.    30
Figure 5: Xylanase crystal after 5 days  0.20 x 0.15 x 0.10 mm    31
Figure 6: Overall structure of Xylanase.    31
Figure 7: Structural alignment of 1ENX(green) and Xylanase from Trichoderma harzianum ETS323(red).    32
Figure 8: Ramachandran plot of xylanase    33
Figure 9: Crystal soaking summary.    34
Figure 10: The xylohexaose electron density in catalytic pocket.(xylotriose_42s data)    35
Figure 11: Xylotriose soaking structure.     35
Figure 12: Xylotriose_3m11s data show only xylotriose density in the catalytic pocket.    36
Figure 13: Compare the substrate position between xylotriose_42s(blue) and xylotriose_3m11s(red)    36
Figure 14: Electrostatic Potential of xylanase complex with xylohexaose.(Xylotriose_42s data)    37
Figure 15: Binding relative residue.    38
Figure 16: Mechanisms of retaining and inverting glycosidases.    38
Figure 17: B-factors compare between naitve and complex structures.    39
Figure 18:  B-factors label. In N-terminus show the higher B-factor and the B-factor of the cord part is low.    40
Figure 19: Xylotriose_35s B-factors label. The section close to residue 107 show higher B-factor than N-Terminus.    40
Figure 20: Pink:From Native structure; Others: From xylotriose_42s; Blue:N; Red:O
Arg122 in xylotriose_42s show orientation change. Arg122 NE atom close to O2 atom help fixing substrate(-1).    41
Figure 21: TLC end product analysis. lane 1 ,15 min incubate; lane 2, 3 min incubate; lane 3, 1 min incubate, Marker use xylotriose and xylopentaose    41
Figure 22: Glycosyl transfer evidence. Arg122, Glu86, Glu177 in subsite (-1).    42
Figure 23: Mechanism of transglycosylation and hydrolysis    43
Figure 24: Binding subsite of GH-11 xylanase.    44

List of Tables
Table 1: Xylanase families properties.    45
Table 2: Native and xylotriose complexes statistics    46
Table 3: Xylopentaose complexes statistics    47
Table 4: Hydrogen bonding interaction between substrate and GH-11 xylanase    48
Table 5: Applications for xylanase    49
Table 6: Kinetic data from GH-11 xylanase (TRX II)    50

                                

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