果蠅的多巴胺乙醯基轉移酶(dopamine N-acetyltransferase, Dat)，是屬於苯烷基胺乙醯轉移酶家族的蛋白酶(EC 2.3.1.87, arylalkylamine N-acetyltransferase, AANAT)，可促使褪黑激素(melatonin)前驅物質的產生，而褪黑激素(melatonin)在生理上的研究，已經被應用在人類的睡眠週期、情緒以及免疫反應當中。
在前期的研究，我們實驗室已成功將多巴胺乙醯基轉移酶(apo form)、多巴胺乙醯基轉移酶/乙醯輔酶A複合晶體(complex form)兩者的蛋白質結構解出，隨後也將多巴胺乙醯基轉移酶和基質間結合的動力學機制進行一連串的分析，更進而成功解出了高達1.20 Å解析度的多巴胺乙醯基轉移酶的三元複合體(多巴胺乙醯基轉移酶/乙醯輔酶A/基質)，我們根據多巴胺乙醯基轉移酶的複合晶體結構得知，乙醯輔酶A是結合於多巴胺乙醯基轉移酶底部的孔洞通道之中，經由等溫滴定微量熱法(ITC)實驗發現到，多巴胺乙醯基轉移酶要先結合輔助因子乙醯輔酶A，才會與基質作用，這個結果顯示輔助因子能夠優先和酵素結合，因此基質只能由酵素頂端的孔洞進入基質接合空腔中。
經由序列比對以及通道運算軟體，發現了兩個位於通道瓶頸中的氨基酸M121以及D142，我們將其分別突變為色胺酸。再經由酵素動力學實驗證實，M121W以及D142W確實會阻礙基質進入疏水性的基質接合空腔，證實了乙醯輔酶A與基質是通過不同的孔洞與多巴胺乙醯基轉移酶結合。經由四種不同的基質的實驗結果發現，多巴胺比較不受到通道阻礙的影響。我們的研究證實了多巴胺乙醯基轉移酶基質通道的存在，也有助於了解果蠅AANAT的通道大小對基質選擇性的影響。

Drosophila melanogaster dopamine N-acetyltransferase (Dat, EC 2.3.1.87) belongs to the arylalkylamine N-acetyltransferase (AANAT) family, which catalyzes the synthesis of the hormone precursor (melatonin). We have solved the structures of Dat in apo form, binary complex (Dat / acetyl coenzyme A) and ternary complex form (Dat / acetylarylalkylamine / CoA) and proposed the catalytic mechanism previously. According to the binding study by isothermal titration calorimetry (ITC), the cofactor (Acetyl-CoA) needed to bind to the Dat prior to substrate, which would hinder the substrate entry to its binding site. Therefore, we speculate that an entry tunnel for substrate may exist to facilitate the substrate binding to the active site. In this study, we replaced two residues with tryptophan, M121 and D142, located inside the tunnel to see the effects of tunnel hindrance. Our DTNB-based enzyme activity measurements and enzyme kinetic studies showed that mutant M121W decreased the enzyme activity and the substrate binding comparing to wild type Dat. Among the four substrates (Dopamine, serotonin, phenylethylamine, tryptamine) tested, only the efficiency of dopamine remains. This result confirms that M121W and D142W may hinder the substrate entry, resulting in decreased binding efficiency of the binary complex. Our studies not only confirm the existence of a substrate tunnel, but also show the tunnel size may contribute to the substrate specificity.

Content
中文摘要 1
Abstract 2
Acknowledgements 3
Content 4
Abbreviations 8
Keywords 9
Chapter 1. Introduction 10
1.1 GCN5-related N-acetyltransferase (GNAT) superfamily 10
1.2 Overview the Arylalkylamine N-acetyltransferase (AANAT) and melatonin 11
1.3 Dopamine N-acetyltransferase (Dat) from Drosophila melanogaster 12
1.4 The aim of this research 13
Chapter 2. Materials and Methods 14
2.1 Construction of Recombinant Drosophila melanogaster Dat 14
2.2 Construction of Drosophila melanogaster Dat mutants 15
2.3 Protein Expression of wild type Dat and mutants 15
2.4 Protein purification of wild type Dat and mutants 16
2.5 Protein concentration 16
2.6 SDS-PAGE 17
2.7 Identification of Dat and Dat mutants molecular weight 17
2.8 Circular Dichroism Spectroscopy 18
2.9 Measurement and determination of the tunnel bottleneck of Dat 18
2.10 DTNB-based function assay (Enzyme Activity Measurements) 19
2.11 Isothermal Titration Calorimetry binding assay 20
Chapter 3. Result and Discussion 21
3.1 Expression and Purification of Dat and ΔDat 21
3.2 The studies of substrate and Cofactor binding to Dat were determined separately via ITC experiments 22
3.3 Substrate tunnel surmise by Dat structure observation 22
3.4 Determining the amino acid around bottleneck of the Dat substrate tunnel 23
3.5 Mutation of M121 and D142 on tunnel bottleneck 24
3.6 DTNB functional assay and Substrate Selectivity of Dat 24
3.7 Isothermal Titration Calorimetry Binding Assay 25
3.7.1 Substrate binding assay of wild type Dat 25
3.7.2 Substrate binding assay of M121W and D142W 26
3.7.3 Molecular model of M121W and D142W 26
Chapter 4. Conclusion 27
List of Tables 29
Table 2.1 Primers sequence and conditions for PCR amplification of Drosophila Dat 29
Table 2.2 The formula of PCR for site-directed mutagenesis 30
Table 2.3 The PCR program for site-directed mutagenesis 31
Table 2.4 The concentration of substrates and proteins for ITC experiments 32
Table 3.1 ITC data for substrate and cofactor titrate with Dat, respectively 33
Table 3.2 Comparison of kinetic constants for selected substrates 34
Table 3.3 Kinetic parameters of wild-type for substrate specificity 35
Table 3.4 Kinetic parameters of M121W for substrate specificity 36
Table 3.5 Kinetic parameters of D142W for substrate specificity 37
Table 3.6 ITC data for titration with wild-type Dat. 38
Table 3.7 ITC data for titration of wild-type or selected tryptophan-substituted forms of Dat with dopamine. 39
Table 3.8 ITC data for titration of wild-type or selected tryptophan-substituted forms of Dat with phenyl-ethylamine. 40
Table 3.9 ITC data for titration of wild-type or selected tryptophan-substituted forms of Dat with tryptamine. 41
Table 3.10 ITC data for titration of wild-type or selected tryptophan-substituted forms of Dat with serotonin. 42
List of Figures 43
Figure 1.1 Topology of the GNAT superfamily fold and structure of binary complex (Dat and Acetyl-CoA) 43
Figure 1.2 Chemical structures of the Dat experimentally tested substrates 44
Figure 1.3 The biochemical pathway and enzymatic reaction for the biosynthesis of melatonin. 45
Figure 1.4 Produce melatonin in the pineal gland of the brain 46
Figure 1.5 Catalytic mechanism of Drosophila Dat 47
Figure 1.6 Structure of binary complexe (Dat and Acetyl-CoA) of Drosophila Dat 48
Figure 1.7 Structure of ternary complexe (Dat/Acetyl-CoA/tryptamine) of Drosophila Dat 49
Figure 1.8 The Structure of apo form SNAT and complexed with a bisubstrate analog (sheep). 50
Figure 1.9 The amino acid sequence comparison of SNAT (sheep) and Dat (Drosophila melanogaster) 51
Figure 2.1 Recombinant vector of the plasmid pGEX-6p3 used for the expression of Dat 52
Figure 2.2 Standard curve of bovine serum albumin (BSA) 53
Figure 2.3 The chemical mechanism of Dat based on DTNB functional assay 54
Figure 3.1 Purification of GST-Dat and its variants by ÄTKA prime system 55
Figure 3.2 Expression and Purification of Dat 56
Figure 3.3 Mass spectra of Dat 57
Figure 3.4 ITC analyses of substrate and cofactor titrate with Dat, respectively 58
Figure 3.5 The overall Structure of the Dat ternary complex 59
Figure 3.6 Acetyl-CoA and substrate entrance of the Dat tunnel 60
Figure 3.7 Overview the tunnel of Dat 61
Figure 3.8 Sequence alignment of insect AANATs 62
Figure 3.9 Observed the internal channel of Dat by the Caver software 63
Figure 3.10 Measure the tunnel of Dat 64
Figure 3.11 Determine the bottleneck amino acid of Dat 65
Figure 3.12 Site-directed mutagenesis of recombinant Dat DNA 66
Figure 3.13 Mass spectra of M121W and D142W 67
Figure 3.14 The circular dichorism spectra of Dat and Dat mutants 68
Figure 3.15 DTNB-based assay: wild type Dat, M121W an D142W 69
Figure 3.16 Catalytic efficiency values of three mutants for acetylation of all arylalkylamine substrates 70
Figure 3.17 ITC profiles of (A) Dopamine, (B) Phenyl-ethylamine, (C) Tryptamine, (D) Serotonin titrate to wild type Dat. 71
Figure 3.18 ITC profiles of (A) wild type, (B) M121W, (C) D142W binding to dopamine 72
Figure 3.19 ITC profiles of (A) wild type, (B) M121W, (C) D142W binding to phenyl-ethylamine 73
Figure 3.20 ITC profiles of (A) wild type, (B) M121W, (C) D142W binding to tryptamine 74
Figure 3.21 ITC profiles of (A) wild type, (B) M121W, (C) D142W binding to serotonin 75
Figure 3.22 Molecular model of M121W 76
Figure 3.23 Molecular model of D142W 77
Figure 3.24 Calculated the tunnel bottleneck of M121W by the Caver software. 78
Figure 3.25 Calculated the tunnel bottleneck of D142W by the Caver software. 79
Reference 80

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