芳烴基烷基胺乙醯基轉移酶 (arylalkylamine N-acetyltransferase, AANAT)是一種將乙醯輔酶A (acetyl-coenzyme A)上的乙醯基轉移到芳烴基烷基胺(arylalkylamine)的一種酵素，而這類的芳烴基烷基胺包含了多巴胺、酪胺、血清胺、苯乙胺等等。AANAT參與了很多生理反應例如調節日夜睡眠週期、骨質生成、單胺的分解等等。在西元2000年以前，果蠅中的AANAT變異型A，也被叫做多巴胺乙醯基轉移酶 (dopamine N-acetyltransferase, Dat)，是唯一一個被確定的AANAT。在2014年時，七個被推測為與AANAT類似的酵素 (AANATL2-AANATL8)被鑑定出來。雖然這些AANATL酵素與Dat之間只有30%序列同一性，但他們仍保有催化乙醯化的反應。AANATL7是其中一種AANATL酵素，在先前的研究中，已被認定不只擁有催化arylalkylamine，也可以催化組胺(histamine)進行乙醯化反應的功能。我們也很好奇AANATL7和Dat的不同。此外，Dat在與乙醯輔酶A結合後，會促使結構改變，形成受質與活化反應的結合位。因此，我們也想調查AANATL7的結構與活性間的關係，這篇研究中，我們試著解出它的晶體結構，找出受質結合、乙醯輔酶A結合、催化相關的關鍵殘基，並將其設計為丙胺酸取代的突變株，並藉由等溫低定量熱法 (Isothermal titration calorimetry, ITC)和艾爾曼試劑 (Ellman’s reagent, DTNB assay)研究突變株對配體結合能力與催化能力的變化。從結果發現，AANATL7如同Dat會先與乙醯輔酶A結合，再和受質結合進行催化反應。從ITC的結果顯示，丙胺酸取代的精胺酸138完全失去了乙醯輔酶A的結合能力，因此精胺酸138對於乙醯輔酶A的結合是相當重要的，而苯丙胺酸22則參與了受質的結合，因為ITC結果可以發現，苯丙胺酸22的突變珠對於乙醯輔酶A的結合能力沒有產生改變，和受質的結合能力卻變得相當弱，同時DTNB assay的結果也顯示，苯丙胺酸22的突變珠無法偵測到乙醯化的產物。而麩胺酸26、蘇胺酸167、絲胺酸171他們扮演著類似Dat之中麩胺酸47、絲胺酸182、絲胺酸186的角色，同樣負責酵素的催化反應。經由ITC、DTNB assay、結構的分析，我們預測麩胺酸26和蘇胺酸167他們作為常見鹼基觸媒，使受質胺基去質子化，產生親核氮原子，開始乙醯化的反應。絲胺酸171則在反應結束後，將輔酶A的硫醇鹽陰離子質子化，使輔酶A穩定。這篇研究提供我們對於果蠅中AANATL7的催化機制與特性。

Arylalkylamine N-acetyltransferase (AANAT) is an enzyme which transfer acetyl group from acetyl coenzyme A (acetyl-CoA) to arylalkylamines substrates. AANAT variant A, also called dopamine N-acetyltransferase (Dat), is the major AANAT in Drosophila melanogaster, which participates in several physical functions including circadian cycle, formation of sclerotin, and breakdown of the monoamine. In 2014, seven putative AANAT-like enzymes (AANATL2-AANATL8) were identified in Drosophila. The sequence identity of AANATL enzymes and Dat is only about 30%, but AANATLs have the similar catalytic abilities of N-acetylation. One of the AANATL enzymes, AANATL7, has been characterized that it could use not only arylalkylamines but also histamine as substrates for N-acetylation. We are interested in the differences between AANATL7 and Dat. In addition, Dat reveals significant conformational change after acetyl-CoA binding, and then results in the formation of substrate binding site and active site. Therefore, we also want to investigate the structure-activity relationship of AANATL7. In this study, we tried crystallization and determination of the AANATL7 structure. The critical residues of AANATL7 participating in acetyl-CoA binding, substrate binding, and catalysis were identified by studies on ligand binding (by ITC) and catalytic ability (by DTNB assay) of their alanine-substituted mutants. In conclusion, AANATL7, like Dat, follows an ordered sequential mechanism with acetyl-CoA binding first and substrate binding afterward. R138 is important for acetyl-CoA binding because the ITC data revealed that mutant R138A almost totally lost acetyl-CoA binding ability. F22 participates in substrate binding because the ITC profile of mutant F22A indicated that no change for acetyl-CoA binding but only a very weak interaction with substrate, while the DTNB assay of F22A showed that no N-acetylation products was detected. E26, T167, and S171 play the similar roles with the catalytic triad in Dat to catalyze the N-acetylation. Combine the ITC data, DTNB assay results, and structural analysis, we propose that E26 and T167 serve as general bases to perform the deprotonation of the amine substrate to result in the nucleophile N atom for N-acetylation. S171 serves as general acid to protonate and stabilize the CoA after the N-acetylation. This study provides us with a better understanding of the enzyme characteristics and catalytic mechanism of AANATL7 in Drosophila melanogaster.

Index
Chapter 1. Introduction 1
1.1 Overview of Arylalkylamine N-acetyltransferase 1
1.2 Dopamine N-acetyltransferase in insects 2
1.3 Arylalkylamine N-acetyltransferase like family 3
1.4 Arylalkylamine N-acetyltransferase like 7 in Drosophila melanogaster 3
1.4.1 Characterization of Arylalkylamine N-acetyltransferase like 7 3
1.4.2 Critical residues of Arylalkylamine N-acetyltransferase like 7 4
1.4.3 Proposed mechanism of Arylalkylamine N-acetyltransferase like 7 4
1.5 Aim of this study 5
Figures of Chapter 1 6
Figure 1.1 The catalytic mechanism of Dat 6
Figure 1.2 The sequence alignment of AANATL family and Dat 7
Figure 1.3 The proposed catalytic mechanism of AANATL7 8
Chapter 2. Material and Methods 9
2.1 Material 9
2.2 Construction of plasmid pAANATL7 10
2.3 Construction of AANATL7 mutants 10
2.4 Protein Expression 11
2.5 Protein Purification 11
2.6 Protein electrophoresis 12
2.7 Identification of molecular weight by Mass spectrometry 12
2.8 Protein quantification 13
2.9 Crystallization condition screening 13
2.10 Determination of Secondary structures by Circular Dichroism (CD) Spectroscopy 14
2.11 Determination of enzyme activity by DTNB assay 15
2.12 Determination of binding affinity by Isothermal Titration Calorimetry (ITC) 15
2.13 Modelling structure and ligand docking of AANATL7 16
Tables and Figures of Chapter 2 17
Table 2.1 The primers of mutated AANATL7 for PCR 17
Table 2.2 Theoretical molecular weights and extinction coefficient of
AANATL7 and mutants 18
Table 2.3 The list of Crystallization condition screening kits 19
Figure 2.1 Plasmid map and protein sequence. 20
Figure 2.2 Chemical reaction diagram of DTNB assay 21
Chapter 3. Result and Disscusion 22
3.1 Expression and purification of L7-WT 22
3.2 Substrate preference of L7-WT 22
3.3 Investigation of the ligands binding and enzyme kinetics of L7-WT 23
3.4 Protein preparation and crystallization condition screening 24
3.5 Selection of critical residue candidates in AANATL7 24
3.6 Expression and purification of AANATL7 mutants 25
3.7 Comparison of the enzyme activity of L7-WT and mutants 26
3.8 Conclusion 29
Tables and Figures of Chapter 3 31
Table 3.1 Kinetic parameters for L7-WT and mutants 31
Table 3.2 Binding affinity and thermodynamic parameters of L7-WT and mutants. 32
Table 3.3 The selected residues in Dat and AANATL7 and their roles in Dat. 33
Table 3.4 Kinetic parameter of Dat and mutants. 34
Figure 3.1 Construction of plasmid pAANATL7. 35
Figure 3.2 Expression and purification of L7-WT. 36
Figure 3.3 Mass spectrum of L7-WT. 37
Figure 3.4 Structures of arylalkylamines substrates. 38
Figure 3.5 Substrate preference of L7-WT. 39
Figure 3.6 Analysis of acetyl-CoA titration and PEA titration into L7-WT by ITC. 40
Figure 3.7 Michaelis-Menten plot of the DTNB assay result of L7-WT. 41
Figure 3.8 Purification of tag-free AANATL7 by gel filtration. 42
Figure 3.9 The suspected crystals in microscope. 43
Figure 3.10 Sequence alignment of AANATL7 and Dat. 44
Figure 3.11 Modelling structure and docking result of AANATL7. 45
Figure 3.12 Analysis of the residues interacted with Acetyl-PEA (in AANATL7
docking model) by Ligplot+. 46
Figure 3.13 Analysis of the residues interacted with CoA (in AANATL7 docking
model) by Ligplot+. 47
Figure 3.14 Structural alignment of AANATL7 docking model and Dat ternary complex. 48
Figure 3.15 Critical residue candidates in AANATL7 docking model. 49
Figure 3.16 Residues may interact with Acetyl-PEA. 50
Figure 3.17 Residues may participate in CoA binding. 51
Figure 3.18 Residues may participate in the catalytic reaction. 52
Figure 3.19 Mutagenesis PCR results of AANATL7 mutants. 53
Figure 3.20 The results of mutagenesis. 54
Figure 3.21 Purification of AANATL7 mutants 55
Figure 3.22 Purification of AANATL7 mutants 56
Figure 3.23 Purification of AANATL7 mutants 57
Figure 3.24 Circular dichorism spectra of L7-WT and mutants. 58
Figure 3.25 Michaelis-Menten plot of the DTNB assay results of L7-WT and its mutants. 59
Figure 3.26 Comparison of ITC data of L7-WT and mutants of acetyl-CoA binding candidates. 60
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