臺灣博碩士論文加值系統

海藻糖合成酶（Trehalose synthase, TS）屬於糖苷鍵水解酶第13家族（Glycoside hydrolase family 13, GH13），催化可逆的異構化反應將麥芽糖轉換成海藻糖。因為這個酵素具有反應步驟簡單，而且反應基質便宜的特性，因此具有被運用於海藻糖之工業生產的潛力。過去，由實驗室王詠霖學長測定出抗輻射奇異球菌（Deinococcus radiodurans）海藻糖合成酶與抑制劑 Tris共結晶的複合晶體結構（DrTS-Tris），顯示了一個因受質結合而形成的封閉構型，可使 TS進行分子內異構化作用。藉由比對 DrTS-Tris以及與其相似的蛋白質結構發現，與活性位-1 subsite的葡萄糖形成交互作用力的氨基酸具有高度保留性；相較之下，活性位 +1 subsite的周遭則有著多樣化與高相異性的氨基酸，因此推測這些氨基酸可能參與調控這些不同酵素的受質與反應特異性。
本篇論文的主要研究目標為探討在 DrTS活性位 +1 subsite周遭氨基酸的功能。首先，王詠霖學長建立了 DrTS與麥芽糖的模擬複合體結構。根據酵素活性分析顯示，Y213A，E320A與 E324A突變株並無偵測到產物的生成，因此推測 Tyr213，Glu320與 Glu324這三個氨基酸對於 DrTS的活性是必須的；然而，N253A突變株則具有野生株 ~12%的異構酶以及 ~163%的水解酶活性。在 DrTS當中，天門冬醯胺酸253為位於 general acid/base谷氨酸之後的兩個氨基酸，並且高度的保留於來自不同物種的 TS序列當中。在一些 GH13成員的突變研究中顯示，這個位置的氨基酸影響了酵素的受質與反應特異性。除此之外，由我測定的 DrTS與海藻糖相似物 validoxylamine A之複合晶體結構可以推測，天門冬醯胺酸253與海藻糖的距離，近於與麥芽糖的距離。因此，我的博士論文為利用飽和點突變，以及分析突變株的酵素活性與蛋白質結構，來了解天門冬醯胺酸253的功能研究。
在所有的天門冬醯胺酸253突變株當中，N253R為唯一一個無法偵測到酵素活性的突變株。其晶體結構顯示，精氨酸253的支鏈伸入活性位，可能因此阻擋了麥芽糖與海藻糖的結合位。此外，有四個突變株：N253A，N253C，N253D與 N253L，具有相對野生株較低的異構酶以及較高的水解酶活性。在 N253A與 N253C之晶體結構的活性位上方觀察到可使水分子通透的孔洞，可能因此導致更多的水分子進入活性位而水解反應中間產物。至於剩下的十四個突變株，則僅具有麥芽糖的水解活性。藉由將 N253Q，N253E與 N253T的蛋白質晶體結構與 DrTS-海藻糖的模擬複合體結構重疊推測，突變的氨基酸分子支鏈可能會與海藻糖在 +1 subsite產生立體空間障礙。因此，這些突變株無法將海藻糖作為受質以及產物。出乎意料的是，N253F突變株的晶體結構顯示了一個新的開放構型。將其與 DrTS相似的結構比較顯示，N253F的結構可能代表 TS於與受質結合前的構型。
綜合上述的實驗結果顯示，輻射奇異球菌海藻糖合成酶的天門冬醯胺酸253已經被演化成為最適合催化分子內異構化作用的氨基酸。這個在 TS當中高度保留的天門冬醯胺酸不僅參與受質引發的活性位關閉外，也調控了反應特異性。

Trehalose synthase (TS) catalyzes a simple conversion of the maltose into trehalose with a side reaction of hydrolysis and hence is of great interest to industrial trehalose production. Dr. Yung-Lin Wang has solved the crystal structure of Deinococcus radiodurans TS in complex with the inhibitor Tris (DrTS-Tris), which represents a substrate-induced closed conformation for catalysis of the intramolecular isomerization. A structural comparison between DrTS-Tris and its closest structural neighbors reveals that residues interacting with the glucose residue at the -1 subsite are conserved, while residues surrounding the +1 subsite are diverse and hence may be responsible for the substrate and reaction specificity.
The specific aim of this thesis is to characterize the residues at the +1 subsite of DrTS. A maltose was first modeled into the active site. Subsequent mutational analysis showed neither detectable isomerase nor hydrolase activity in Y213A, E320A and E324A mutants, suggesting that these residues are essential for the TS activity. In contrast, replacement of Asn253 with alanine resulted in ~12% of isomerase activity and ~163% of hydrolase activity compared with the wild type. This strictly conserved asparagine residue is the second residue behind the general acid/base Glu, in which the corresponding residue in some other glycoside hydrolase family 13 (GH13) enzymes has been shown to participate in the substrate and reaction specificity. In addition, I solved the complex structure of DrTS with a trehalose analogue, validoxylamine A, displaying that Asn253 forms closer contacts with trehalose than maltose. Thus, functional roles of Asn253 were characterized by saturation mutagenesis and structural analysis.
N253R is the only mutant showing no detectable enzyme activity, and the crystal structure reveals that the protruding guanidino side chain blocks the substrate binding site. Four mutants including N253A, N253C, N253D and N253L, contain a reduced isomerase activity and retain an increased hydrolase activity. The N253A and N253C structures demonstrate a mutant-induced active-site aperture for water entry. The remaining fourteen mutants possess only maltose hydrolase activity. The N253Q, N253E and N253T structures suggest that these substituted side chains make potential steric hindrance with the glycosyl moiety of trehalose at the +1 subsite and hence trehalose can serve as neither substrate nor product. Unexpectedly, the N253F mutant structure reveals a novel open TS conformation with an empty active site, in which the closed active conformation may not formed upon substrate binding. A comparison with the closest structural matches of TS suggests that this empty active-site topology represents an open conformation for the apo enzyme prior to substrate binding.
In summary, the saturated mutation demonstrated that the Asn253 in DrTS has been evolved to be the most appropriate residue for catalysis of the isomerzation reaction. This residue not only participates in the substrate-induced active-site closure through hydrogen bonding with Glu324, but also determines the maltose/trehalose preference and the isomerase/hydrolase preference. Hopefully, this study can provide new insights into the protein engineering of TS for potential industrial applications.

中文摘要....i
Abstract....iii
Table of Contents....v
List of Tables....vii
List of Figures....viii
List of Abbreviations....ix
Chapter 1 Introduction
1.1 Trehalose....1
1.1.1 Biological functions....1
1.1.2 Properties and applications....2
1.1.3 Biosynthetic pathways....3
1.1.4 Industrial production....5
1.2 Trehalose synthase (TS)....7
1.2.1 Enzyme properties....7
1.2.2 A kinetically proposed catalytic mechanism....7
1.2.3 Structural studies....8
1.3 Glycoside hydrolase family 13 (GH13)....9
1.3.1 A comparison between DrTS and some other GH13 enzymes....10
1.3.2 The conserved -1 subsite and the diverse +1 subsite....12
1.3.3 Substrate-induced formation of the active conformation....13
1.4 Aims of this study....14
Chapter 2 Materials and Methods
2.1 Chemicals....15
2.2 Protein preparation and characterization....16
2.2.1 Site-directed mutagenesis....16
2.2.2 Protein expression and purification....16
2.3 Activity assay....17
2.4 Validoxylamine A (VAA) preparation....18
2.5 Crystallization....19
2.6 X-ray diffraction data collection and structure determination....20
Chapter 3 Results and Discussion
3.1 Crystal structure of DrTS-VAA complex....21
3.2 Rationale for saturation mutagenesis of Asn253....22
3.3 Enzymatic activity of Asn253 mutants....24
3.4 Crystal structures of Asn253 mutants....26
3.4.1 N253R: Blocking of the substrate binding site....27
3.4.2 N253A, N253C and R148A: A mutation-induction of an active-site aperture....27
3.4.3 N253Q, N253E, N253T and N253H: A potential steric hinderance with trehalose....28
3.4.4 N253F and N253RD: A novel open conformation and its implications....30
3.4.4.1 A snapshot of the apo-TS....31
3.4.4.2 The open conformation confers the hydrolytic activity to N253F....32
3.5 The proposed catalytic mechanism....33
References....36
Tables....51
Figures....57
Appendices....84

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