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基轉移酶家族中的蛋白酶(EC 2.3.1.87, Arylalkylamine N-Acetyltransferase, AANAT)，它們可以催化乙醯輔酶A (Acetyl-CoA) 的乙醯基團轉移至苯烷基胺上，而產出N-乙醯苯烷基胺。近期以來，我們實驗室已經解出多巴胺乙醯基轉移酶晶體、多巴胺乙醯基轉移酶/乙醯輔酶A複合晶體兩者之蛋白質結構。隨後更進一步解出解析度高達1.2 Å的多巴胺乙醯基轉移酶的三元複合晶體 (乙醯基轉移酶/乙醯基輔酶A/基質) 的蛋白質結構。藉由此三元複合體的結構加上先前實驗室所做的等溫滴定微量熱法實驗，我們發現乙醯基轉移酶需要先結合乙烯基輔酶A，才能夠與基質作用。因此我們發現基質與乙醯基輔酶A進入乙醯基轉移酶的入口是不相同的。在先前的研究，我們實驗室發現M121以及D142坐落於基質通道的最狹窄處 (通道瓶頸)。當M121以及D142分別突變成色胺酸，經由酵素動力學實驗發現M121W以及D142W會阻礙基質進入到疏水性的基質結合空腔，進而證實通道的存在。在本實驗中，我們將M121以及D142分別突變成丙氨酸，使得通道瓶頸的直徑變大。另外也將M121以及D142一併突變成色胺酸，使得通道瓶頸變得比個別突變還小。經由酵素動力學的實驗，我們發現讓通道瓶頸變寬的D142A，與wt-Dat有著幾乎相同的活性，且對不同大小的測試基質的選擇性不如wt-Dat顯著。而讓通道瓶頸變更窄的M121WD142W的酵素活性則是有顯著降低的現象，從這結果我們可以發現通道瓶頸的大小確實會影響酵素的活性。然而，M121A雖然使得通道瓶頸變大，卻造成酵素活性顯著降低。使用LIGPLOT和PyMol進行結構分析後，我們發現M121對基質可以產生S/π作用，所以當M121被改變成丙氨酸時，改變得不單純只是通道瓶頸的大小，而是直接影響基質結合。

Drosophila dopamine N-acetyltransferase (Dat, EC 2.3.1.87) is an arylalkylamine N-acetyltransferase (AANAT), which can catalyze acetyl transfer from acetyl-coenzyme A to arylalkylamine, yielding N-acetylarylalkylamine. According to our X-ray structure, a tunnel was inside the protein with entrances at top and bottom. It has also been shown that the substrate located at the middle of Dat, and CoA located at the bottom which seemed to completely block the entrance. Dat exhibits an ordered sequential mechanism, with acetyl-CoA binding first, followed by substrate. Therefore, substrate should access to its binding site through the other entrance, and it looked like a tunnel existed. M121 and D142 located in the narrowest site of the tunnel (tunnel bottleneck). The replacement of these two residues with tryptophan resulted in a decrease in enzyme activity which may imply the hindrance to the substrate entrance by M121W and D142W. In the present study, the size effect of the substrate tunnel was on enzyme activity. We replaced M121 and D142 to alanine individually, which cause the tunnel bottleneck broader. We also made a double mutation, M121WD142W, to make the tunnel bottleneck even narrower than single mutation. The enzyme kinetic studies demonstrated that D142A had almost the same enzyme activity as that of wild type, and M121WD142W showed a significant decrease in the enzyme activity compared to wild type, M121W and D142W. These results confirmed that the size of tunnel bottleneck may affect the substrate specificity again. Although the tunnel bottleneck is broader, M121A exhibits a decrease in enzyme activity. After analysis by LIGPLOT and PyMol, we found that M121 should participate in substrate binding via S/π interactions. Therefore, changing M121 to alanine not only change the size of substrate tunnel bottleneck, but also was expected to significantly decrease the substrate binding.

中文摘要 2
Abstract 3
Content 4
Abbreviations 7
Keywords 8
Chapter 1. Introduction 9
1.1 Dopamine N-acetyltransferase (Dat) as an AANAT from Drosophila melanogaster 9
1.2 The overall structure of Drosophila Dopamine N-acetyltransferase 10
1.3 Substrate-entry tunnel of Dopamine N-Acetyltransferase 11
1.4 Motivation of the study 12
Chapter 2. Materials and Methods 13
2.1 Measurement and determination of the tunnel size of Dat 13
2.2 Protein Expression and Purification 13
2.2.1 Protein construction, Recombinant and Mutagenesis of Dat 13
2.2.2 Protein Expression of wild type Dat and mutants 14
2.2.3 Protein purification of wild type Dat and mutants 15
2.3 SDS-PAGE 15
2.4 Quantification of protein concentration 16
2.5 Identification of protein purity and molecular weight 16
2.6 Circular Dichroism Spectroscopy 17
2.7 Isothermal Titration Calorimetry binding assay 18
2.8 DTNB-based function assay 18
Chapter 3. Results and Discussion 20
3.1 Designing the mutants and measuring the changes in simulated mutants structures 20
3.2 Expression and Purification of wild-type Dat (wt-Dat) and its mutants 20
3.3 Secondary structures of wt-Dat and its mutants 21
3.4 Isothermal Titration Calorimetry Binding Assay 21
3.5 DTNB-based functional assay 23
Chapter 4. Conclusion 25
List of Tables 26
Table 2.1 Primer sequences and conditions for PCR amplification of Drosophila Dat 26
Table 3.1 ITC data for titration of wild type Dat and its mutants with dopamine 27
Table 3.2 Kinetic parameters of wild type Dat 28
Table 3.3 Kinetic parameters of D142A 29
Table 3.4 Kinetic parameters of M121WD142W 30
Table 3.5 Kinetic parameters of M121A 31
Table 3.6 Comparison of the relative enzyme activity of wild type and mutants 32
Table 3.7 Comparison of ITC data with M121W and D142W 33
List of Figures 34
Figure 1.1 The enzymatic reaction and biochemical pathway for the biosynthesis of melatonin. 34
Figure 1.2 The pleiotropic effect of melatonin 35
Figure 1.3 Emotional competence rhythms regulate by neurotransmitters in brain 36
Figure 1.4 Topology of GNATs superfamily and the binary complex structure of Dat (Dat/Acetyl-CoA) 37
Figure 1.5 The catalytic mechanism of Dat. 38
Figure 1.6 The binary complex structure of Dat (Dat/Acetyl-CoA) 39
Figure 1.7 The overall structure of the ternary complex of Dat 40
Figure 1.8 Acetyl-CoA and substrate entrances of Dat tunnel 41
Figure 1.9 The tunnel size of wild type Dat calculated by Caver software 42
Figure 1.10 Sequence alignment of insect AANATs 43
Figure 2.1 Map of the plasmid pGEX-6p3 used for the characterization of Dat 44
Figure 2.2 The catalytic reaction of AANAT based on DTNB functional assay 45
Figure 3.1 The sizes of tunnel bottleneck in M121A and D142A calculated by Caver software 46
Figure 3.2 The sizes of tunnel bottleneck in M121WD142W calculated by Caver software 47
Figure 3.3 Purification results of wild type Dat via ÄTKA prime system 48
Figure 3.4 Expression and purification of wt-Dat monitored by 13% SDS-PAGE 49
Figure 3.5 Expression and purification of mutants monitored by 13% SDS-PAGE 50
Figure 3.6 Mass spectra of mutants 51
Figure 3.7 The circular dichroism spectra of Dat and mutants 52
Figure 3.8 ITC profiles of (A) wild type Dat, (B) M121A, (C) D142A and (D) M121WD142W binding to dopamine. 53
Figure 3.9 S/π interactions between M121 and substrates of Dat 54
Figure 3.10 The potential association between Tyr64, Lys144 and Asp142 55
Figure 3.11 Catalytic efficiency of wild type Dat and its mutants 56
Figure 3.12 DTNB-based functional assay: wt-Dat, M121A, D142A and M121WD142W 57
Figure 3.13 The influence of the substrate entrance tunnel of D142A and D142W 58
Appendix I: DTNB-based functional assay of M121W and D142W 59
Appendix II: ITC data for titration of WT, M121W and D142W with dopamine. 60
Appendix III: DTNB-based functional assay of Y64A 61
Appendix IX: ITC data for titration of Y64 with dopamine, 62

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