乙醯輔酶A在昆蟲芳烷基胺N-乙醯基轉移酶催化循環中蛋白構象調節的重要作用

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研究生：	吳竹雅 Wu, Chu-Ya
論文名稱：	乙醯輔酶A在昆蟲芳烷基胺N-乙醯基轉移酶催化循環中蛋白構象調節的重要作用 An essential role of acetyl coenzyme A in the regulation of protein conformation of insect arylalkylamine N-acetyltransferase (AANAT) in the catalytic cycle
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:	徐尚德 Hsu, Shang-Te Danny 莊偉哲 Chuang, Woei-Jer 蘇士哲 Sue, Shih-Che 鄭惠春 Cheng, Hui-Chun
學位類別：	博士 Doctor
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：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	117
中文關鍵詞：	芳烷基胺N-乙醯基轉移酶、乙醯輔酶A 、多巴胺N-乙醯基轉移酶
外文關鍵詞：	arylalkylamine N-acetyltransferase, acetyl coenzyme A, dopamine N-acetyltransferase
相關次數：	點閱：2 下載：0
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多巴胺N-乙醯基轉移酶（Dat）是一種芳烷基胺N-乙醯基轉移酶（AANAT）。在昆蟲中，AANAT扮演多個重要生理功能，例如神經傳導物質去活化、昆蟲的外骨骼發育與晝夜節律。AANAT通過次順序結合機制進行N-乙醯基催化反應，其中輔因子（乙醯輔酶A，Ac-CoA）先結合，然後受質（單胺類，如:多巴胺、色胺）再結合。乙醯基轉移後，新的Ac-CoA可以釋放Dat中的產物（CoA和乙酰基產物）。然而，在催化循環過程中，AANAT的蛋白結構做了什麼調控，哪些殘基參與構象調節，進而影響結合機制與催化機制尚不清楚。在本研究，我們透過單晶繞射和水溶液核磁共振光譜分析了Dat結構：單元形式、結合Ac-CoA的二元形式和Dat/CoA/乙醯基產物的三元形式，並探討了催化循環中每個階段Dat的結構變化。結果顯示，Ac-CoA的結合誘導了Dat蛋白構形的轉變，使構形從開放狀態變為關閉狀態，形成了受質結合口袋與催化口袋。此外，催化口袋的形成也依賴Ac-CoA上的乙醯基團來微調。同時，Ac-CoA的結合也縮小了Dat反應通道的閘門，創造了反應通道的瓶頸。我們利用結晶學與NMR觀察產物的釋放。結果顯示，新的Ac-CoA可以釋放Dat三元複合物中的產物（CoA和乙酰基產物），其中，乙醯基產物的釋放需要通過Met121與Asp142圍繞的瓶頸，Met121的側鏈可能作為一扇門去調控產物釋放。
最後，本研究使用Asp46的丙氨酸突變來驗證鹽橋對於Dat結構調控的貢獻。我們的數據表明，受質結合口袋與反應通道瓶頸的形成，依賴Asp46-Arg153的鹽橋作用力來做為Dat結構轉換的開關。Asp46-Arg153鹽橋的作用力也增加了α1區域與受質入口的剛性。綜上所述，本研究從結構的角度提供AANAT蛋白在催化循環過程中的重要調控機制，未來可以做為一個模型基礎應用在其他昆蟲AANATs或脊柱動物AANATs的系統。

Dopamine N-acetyltransferase (Dat) is an arylalkylamine N-acetyltransferase (AANAT). In insects, AANAT plays many important roles in physiological functions, such as inactivation of neurotransmitters, development of insect exoskeleton, and circadian rhythm. AANAT catalyzes N-acetylation through an ordered sequential mechanism in which cofactor (acetyl coenzyme A, Ac-CoA) binds first followed by substrate (monoamines, such as dopamine and tryptamine) binding. After acetyl transfer, a new Ac-CoA can release the products (CoA and acetyl-product) from Dat. Notwithstanding, research on the protein conformational regulatory of AANAT in progressing the catalytic cycle has been scarce and awaits elucidation. In this study, we analyzed the structures of Dat in apo form, Ac-CoA binary form, and Dat/CoA/acetyl-product ternary form by crystallography and solution NMR, and addressed structural changes of Dat at each stage in the catalytic cycle. The results showed that the binding of Ac-CoA induced the conformational change of Dat from the open state to the closed state, which formed a substrate binding pocket and a catalytic pocket. Furthermore, the catalytic pocket also relied on the acetyl group of Ac-CoA to fine-tune the pocket. Meanwhile, the binding of Ac-CoA also narrowed the gate and created a bottleneck in the reaction tunnel of Dat. We used X-ray crystallography and nuclear magnetic resonance (NMR) to monitor the release of the products. It showed that the new Ac-CoA can release the products (CoA and acetyl-product) in the Dat ternary complex. The released acetyl-product needed to pass through the bottleneck surrounded by Met121 and Asp142, and the side chain of Met121 could act as a wing gate to control the product release.
Finally, in this study the alanine mutation of Asp46 was used to verify the contribution of salt bridges to the regulation of Dat structure. Our data showed that the formation of the substrate binding pocket and the bottleneck of the reaction channel relied on the salt bridge interaction of Asp46-Arg153 to switch the structure of Dat. The salt bridge, Asp46-Arg153, also increased the rigidity of the α1 region and the substrate entrance. In summary, this study provides a structural basis for the regulatory mechanism of the AANAT protein in the catalytic cycle, which can be used as a raw model of other insect AANATs or vertebrate AANATs in the future.

中文摘要    1
Abstract    3
Acknowledgements    5
Content    6
List of Tables    9
List of Figures    10
Abbreviations    13
Chapter I. Introduction    15
1 Physiological functions of arylalkylamine N-acetyltransferase (AANAT)    15
2 AANAT as a member of GCN5-related N-acetyltransferase (GNAT) superfamily    17
3 Conformational changes and substrates recognition in AANAT and GNAT protein    18
4 Structural feature of insect AANATs    19
5 Catalytic mechanism of AANATs    19
6 Motivation of this study    21
Chapter II. Materials and Methods    23
1 Protein expression and purification    23
2 Crystallization and data collection    24
3 Structure determination    25
4 NMR resonance assignments    25
5 Amino acid selective unlabeling used in identifying resonance assignments    26
6 NMR titration experiments    28
7 Isothermal titration calorimetry (ITC) experiment    28
8 Differential scanning calorimetry (DSC)    29
9 Circular dichroism (CD) spectroscopy    30
Chapter III. Results    31
1 Characterization of Dat    31
2 NMR assignment of Dat in apo, binary, and ternary complexes    32
3 Chemical shift predicted secondary structure of Dat    34
4 Chemical shift differences revealed a conformational change of Dat from initial stage to cofactor binding stage    35
5 Structural basis for Dat substrate binding    37
5.1 Substrate binding pocket    37
5.2 Cofactor binding pocket and the catalytic site    38
6 Overall structural comparison of Dat in each stage of catalysis    39
6.1 Overall structures    39
6.2 The closed form of the cofactor binding pocket    40
6.3 Catalytic site    40
6.4 Substrate entrance and binding pocket    41
7 The distinctive roles of the acetyl group and the CoA moiety    43
8 Ac-CoA binding is required for the removal of products from Dat    43
9 A gate in the tunnel-shaped cavity of Dat    46
10 The salt bridge Asp46-Arg153 is a structural switch that forms the substrate binding sites    47
11 The salt bridge Asp46-Arg153 can enhance the rigidity of the substrate binding sites    51
Chapter IV. Discussion    53
1 Catalytic cycle of Dat and the structural regulation    53
2 The structural switch couples the cofactor binding to the formation of substrate binding pocket    53
3 The fine adjustments of the catalytic site    55
4 The role of the tunnel-shaped cavity    56
5 The release of products    58
6 The catalytic mechanism    59
7 Contributions to AANATs and perspective    60
7.1 Conserved salt bridge in AANATs that may modulate substrate binding ability    60
7.2 Investigations of potential inhibitors which are specific for insect AANATs    61
Chapter V. Conclusions    62
Publication list    107
Appendix    108
Reference    114




                                

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