探討岩藻糖轉化酶的催化反應：分子動力學與定點突變模擬分析

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研究生：	林柏全 Lin, Po-Chuan
論文名稱：	探討岩藻糖轉化酶的催化反應：分子動力學與定點突變模擬分析 Exploring the Reaction of Fucosyltransferases: Insights from Molecular Dynamics and In-silico Mutagenesis
指導教授：	楊自雄 Yang, Tzuhsiung
口試委員:	游景晴 Yu, Ching-Ching 林俊宏 Lin, Chun-Hung
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2024
畢業學年度：	113
語文別：	英文
論文頁數：	94
中文關鍵詞：	分子動力學、岩藻糖轉化酶、岩藻糖、定點突變、分子對接
外文關鍵詞：	Molecular dynamics, Fucose, Fucosyltransferase, Mutagenesis, Molecular docking
相關次數：	點閱：36 下載：0
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岩藻糖基化是一種重要的糖基化過程，其涉及岩藻糖向寡糖分子的酶促轉移，該反應由岩藻糖基轉移酶催化。本研究主要在研究兩種岩藻糖基轉移酶——FucTa和Bf13FT，它們在人體母乳和其他體液中合成重要的岩藻糖基化寡糖分子中起著關鍵作用。由於缺乏可用的晶體結構，我們採用了AlphaFold2來預測FucTa和Bf13FT的三維結構，並基於這些預測結構進行了分子對接研究，探索了這些酶與各種寡糖分子之間的結合互作，包括了Lacto-N-tetraose (LNT)和Lacto-N-neotetraose (LNnT)以及不同的linker修飾。
為了進一步深入理解，我們對對接複合物進行了分子動力學(MD)模擬，揭示了這些酶-寡糖分子之間的動態模擬和穩定性。透過這些模擬，我們發現了關鍵胺基酸，特別是FucTa中的GLU-95和Bf13FT中的GLU-75，在與特定糖鏈位置(I3和III3/4)形成氫鍵方面起著至關重要的作用。為了增強這些互作，我們進行了電腦模擬突變，生成了一系列旨在改善寡糖分子結合親和力的突變體。我們對這些突變體進行了額外的對接和MD模擬，以評估其穩定性和互作模式。
研究結果顯示，某些突變可以有效增強所需的酶-寡糖分子的關鍵氫鍵，而另一些突變可能會干擾這些關鍵氫鍵。這些關於岩藻糖基化分子機制的研究不僅推進了我們FucTa和Bf13FT對糖基轉移酶功能的理解，還為設計改進的糖基化過程提供了理論框架。

Fucosylation, a key glycosylation process, involves the enzymatic transfer of fucose to glycan substrates, a reaction catalyzed by fucosyltransferases. In this study, we focus on two fucosyltransferases, FucTa and Bf13FT, which are both reported to involve in the synthesis of important fucosylated glycans found in human milk and other biological fluids. Due to the lack of available crystal structures, we employed AlphaFold2 to predict the 3D structures of FucTa and Bf13FT with high accuracy. These predicted structures served as the structures for molecular docking studies, where we investigated the binding interactions between the enzymes and various glycan substrates, including Lacto-N-tetraose (LNT) and Lacto-N-neotetraose (LNnT), along with different linker modifications.
To further refine our understanding, we conducted molecular dynamics (MD) simulations on the docked complexes, revealing the dynamic behavior and stability of these enzyme-glycan interactions. Through these simulations, we identified critical residues, particularly GLU-95 in FucTa and GLU-75 in Bf13FT, that play a crucial role in forming hydrogen bonds with specific glycan positions (I3 and III3/4). To enhance these interactions, we performed in-silico mutagenesis, generating a series of mutants aimed at improving the binding affinity of the glycan substrates. These mutants were subjected to additional docking and MD simulations to assess their stability and interaction patterns.
Our findings demonstrate that certain mutations can enhance the desired enzyme-substrate interactions, while others may disrupt them. These insights into the molecular mechanisms of fucosylation not only advance our understanding of glycosyltransferase function but also provide a framework for designing improved glycosylation processes.

Abstract    ii
摘要    iii
Table of Contents    iv
List of Figures    vi
List of Tables    xix
Chapter 1.    Introduction and Background    1
1.1.    Fucosyltransferases Bf13FT/FucTa and glycans Lacto-N-tetraose/Lacto-N-neotetraose (LNnT)    2
1.2.    Previous studies and fucosylation site selectivity    4
1.3.    Alphafold2    6
1.4.    Molecular Docking    8
1.5.    Molecular dynamics (MD) simlation    12
Chapter 2    15
Investigation of the interactions between glycans LNT and LNnT and wild type fucosyltransferases FucTa and Bf13FT    15
2.1. Abstract    16
2.2.    Results and Discussion    17
2.2.1.    Generating protein and ligand structures    17
2.2.2.    Molecular docking of LNT and LNnT ligands to wild type FucTa and Bf13FT        20
2.2.3.    Methods of molecular dynamics simulations  of the selected docking modes        22
2.2.4.    Results and analysis of molecular dynamics simulations    23
2.3.    Conclusion    29
Chapter 3  In-silico mutagenesis of fucosyltransferases FucTa and Bf13FT and the interactions between glycans LNT and LNnT and mutants    31
3.1.    Abstract    32
3.2.    Results and Discussion    32
3.2.1.    Selection of the mutants    32
3.2.2.    Performing in-silico mutagenesis of FucTa and Bf13FT    34
3.2.3.    Molecular docking of LNT and LNnT ligands to mutated FucTa and Bf13FT        35
3.2.4.    Molecular dynamics simulations methods of the selected docking modes        39
3.2.5.    Results and analysis of molecular dynamics simulations    39
3.2.6.    The effects of the mutated residues and analysis    53
3.3.    Conclusion    55
Appendix    57
Reference    87


                                

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