果蠅多巴胺乙醯轉移酶（Dopamine N-acetyltransferase, Dat）是一種苯烷基胺乙醯轉移酶（AANAT, EC 2.3.1.87），屬於GCN5相關的胺基端乙醯化轉移酶（GCN5-related N-acetyltransferase, GNAT）超家族中的一類酵素。果蠅多巴胺乙醯轉移酶能催化乙醯化（N-acetylation）反應的進行，將乙醯輔酶A（Acetyl-CoA, Ac-CoA）上的乙醯基（Acetyl group）轉移至苯烷基胺（Arylalkylamine）這類受質的胺基，最終產生乙醯苯烷基胺（N-acetyl-arylalkylamine）和輔酶A。在過去的文獻裡，經由酵素動力學抑制實驗證實苯烷基胺乙醯轉移酶具有定序二二連續型機制（Ordered bi bi sequential mechanism）的酵素特性。在先前的研究中，我們也已經利用等溫滴定量熱儀（Isothermal titration calorimetry, ITC）與酵素動力學抑制實驗（Enzyme inhibition kinetics）證實果蠅多巴胺乙醯轉移酶具有定序二二連續型機制的酵素特性：果蠅多巴胺乙醯轉移酶必須先和乙醯輔酶A結合，才能與受質結合，最終發生乙醯化反應。然而，此機制其中的蛋白質結構資訊尚有不明。此外，在之前實驗室利用浸泡式結晶（Soaking）的方式取得果蠅多巴胺乙醯轉移酶三元複合體（Ternary form）的結構中，發現在輔因子與受質結合位中存在乙醯苯烷基胺和輔酶A的電子雲；在ITC結果，我們發現當果蠅多巴胺乙醯轉移酶結合輔酶A時，會與受質的產物發生結合。由此看來，這兩種產物似乎無法自動釋出酵素外。因此，我們好奇果蠅多巴胺乙醯轉移酶是如何進行酵素回收和催化反應。
在本研究中，我們利用共結晶（Co-crystallization）的方式解出解析度為1.20 Å的果蠅多巴胺乙醯轉移酶三元複合體（tDat/CoA/Ac-PEA complex）結構。比較之前實驗室解出的果蠅多巴胺乙醯轉移酶（Apo form）、二元複合體（tDat/Ac-CoA complex）與本研究解出的三元複合體（tDat/CoA/Ac-PEA complex）結構，我們發現果蠅多巴胺乙醯轉移酶的構型不同於二元複合體與三元複合體；二元複合體與三元複合體的構型是非常相像的。然後，我們進一步發現當乙醯輔酶A結合果蠅多巴胺乙醯轉移酶之後，會引發此酵素構型上的轉變。其中，果蠅多巴胺乙醯轉移酶受質結合位構型的轉變可能是決定受質是否可以結合到此酵素的關鍵因素。因此，根據本研究的結構分析結果提供關於定序二二連續型酵素機制在蛋白質結構上的資訊。此外，比較共結晶與浸泡式結晶所解出的果蠅多巴胺乙醯轉移酶三元複合體結構，可以發現大體結構並無太大的差異，但是在共結晶的三元複合體結構中，蛋白質表面會額外多出一個乙醯苯乙胺（N-acetyl-2-phenylethylamine, Ac-PEA）。這個現象暗示也許有一些因子可以協助果蠅多巴胺乙醯轉移酶產物釋出與酵素循環。透過等溫滴定量熱儀與共結晶所設計的競爭實驗，我們可以發現對於果蠅多巴胺乙醯轉移酶而言，乙醯輔酶A對於輔酶A有較強勢的競爭關係。而這樣一個不對等的競爭關係，可能是導致果蠅多巴胺乙醯轉移酶能夠回收再利用的主要因素。最後，為了瞭解果蠅多巴胺乙醯轉移酶的過渡態（Transition state），我們根據之前實驗室所提出催化三聯體（Catalytic triad）的催化機制中，設計了三個與穀胺酸47（E47）相關的點突變體（E47D、E47Q與E47N）。經由等溫滴定量熱儀與酵素活性實驗，我們可以發現對穀胺酸47進行點突變的三個點突變體相對於野生型（Wild-type）果蠅多巴胺乙醯轉移酶，在與受質的結合親和力與催化活性上都有明顯下降的趨勢。
根據本研究的實驗結果，我們提出了關於果蠅多巴胺乙醯轉移酶的催化循環（Catalytic cycle）假說：果蠅多巴胺乙醯轉移酶需要先與乙醯輔酶A結合引發構型轉變後，才可以與受質發生結合進行乙醯化反應；反應結束後的產物並不會從酵素中自動釋出，而是需要乙醯輔酶A的幫助，將輔酶A與乙醯苯烷基胺從酵素中競爭下來，使酵素回到二元複合體（tDat/Ac-CoA complex）的型態，以利酵素進行下一輪的催化反應。

Dopamine N-acetyltransferase (Dat) found in Drosophila melanogaster belongs to arylalkylamine N-acetyltransferase (AANAT, EC 2.3.1.87) family, which is a member of GCN5-related N-acetyltransferase (GNAT) superfamily. Dat catalyzes arylalkylamine N-acetylation which transfers acetyl group of acetyl-CoA (Ac-CoA) to arylalkylamine to generate N-acetyl-arylalkylamine and CoA. AANAT had been reported the ordered bi bi sequential mechanism by enzyme inhibition analysis as well. In our previous study, we had also determined Dat is ordered bi bi sequential mechanism using isothermal titration calorimetry (ITC) and enzyme inhibition kinetics. Dat has to bind cofactor (Ac-CoA) first and then followed by substrate (Arylalkylamine). Nevertheless, the underlying structural mechanism still remains ambiguous. Furthermore, we had found the electron density map of products, N-acetyl-arylalkylamine and CoA, on substrate and cofactor binding site in ternary structure by soaking. It seemed products cannot auto-release. Thus, we interested in how Dat conducts enzymatic recycling and the catalytic process.
In this study, we solved 1.20 Å resolution ternary structure (Dat/N-acetyl-arylalkylamine/CoA) of Dat by co-crystallization. Comparing apo form, binary form (tDat/Ac-CoA complex) and ternary form (tDat/CoA/Ac-PEA complex) of tDat, we found conformation of apo form Dat was different from binary and ternary form among them; structures of binary and ternary form were similar with each other. Then, we found the conformational change after Ac-CoA binding with tDat. The conformational change of substrate binding site may decide whether tDat can binding substrate or not. Thus, we elucidated ordered bi bi sequential mechanism of Dat by x-ray structural analysis. Additionally, the overall ternary structure of co-crystallization was similar with soaking except for an additional Ac-PEA outside the protein surface. The phenomenon implied may exist some factors to facilitate product release and enzyme recycle of Dat. Using isothermal titration calorimetry (ITC) and x-ray co-crystallization to carry out the competitive experiments, we found Ac-CoA showed dominantly competitive relation with respect to CoA. The dominantly competitive relation between Ac-CoA and CoA may resulted in enzymatic recycle of Dat. Finally, we based on catalytic triad to generate three variants, E47D, E47Q and E47N, for approaching transition state. All of them lost their substrate binding affinity and led to dramatic catalytic activity decrease.
According to our results, we suggested Ac-CoA priorly binds to Dat providing a conformation change which facilitates substrate binding and forms ternary form. Ac-CoA may serve as driving force in catalyzation process of Dat. Our results implied Ac-CoA drives CoA and N-acetyl-arylalkylamine in Dat (ternary form) away, and occupies the cofactor binding site of Dat. Then Dat returns to state of binary form and accomplishes enzymatic recycling.

中文摘要 1
Abstract 3
謝誌 5
Contents 6
List of Tables 8
List of Figures 9
Appendix 11
Abbreviations 12
Chapter 1. Introduction 14
1.1 Arylalkylamine N-acetyltransferase (AANAT ) family 14
1.2 Physiological functions of Dat 14
1.3 GCN5-related N-acetyltransferase (GNAT) superfamily and Ordered sequential bi
bi mechanism 15
1.4 Structure of Dat 16
1.5 Aim 17
Chapter 2. Materials and Methods 18
2.1 Dopamine N-acetyl transferase (Dat) expression and purification 18
2.1.1 Expression and purification of Dat 18
2.1.2 Tricine SDS-PAGE 19
2.2 A280 of proteins quantification 19
2.3 Secondary structure analysis of circular dichroism (CD) Spectroscopy 20
2.4 Binding affinity assay using isothermal titration calorimetry (ITC) 21
2.5 Enzymatic activity determination by DTNB assay 22
2.6 X-ray crystallography 22
2.6.1 Protein Crystallization 22
2.6.2 Experimental data collection and refinement statistics 24
Chapter 3. Results 25
3.1 Ordered Bi Bi Sequential Mechanism of Dat 25
3.2 Catalytic process and enzymatic recycling of Dat 26
3.2.1 Acetyl CoA revealed dominantly competitive relationship toward CoA in Dat 26
3.2.2 Validate the role of acetyl CoA by x-ray crystallography 29
3.3 Three site-directed mutations based on catalytic triad of Dat to
approach transition state 31
3.3.1 Biophysical characteristics of mutations in comparison with wild type tDat 31
3.3.2 Structures of mutations 31
Chapter 4. Conclusion 33
Chapter 5. Discussion 34
Reference 98

1.Hintermann E, Grieder NC, Amherd R, Brodbeck D, and Meyer UA. Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut. Proceedings of the National Academy of Sciences 1996; 93(22):12315–12320.
2.Hintermann E, Jeno P, Meyer UA. Isolation and characterization of an arylalkylamine N-acetyltransferase from Drosophila melanogaster. FEBS Letters 1995;375(1-2):148-50.
3.Bruno Luis Alves Lourenço, Maicon Vinícius Araújo Santos Silva, Elisson Barros de Oliveira, Wagner Rodrigues de Assis Soares, Aristóteles Góes-Neto, Gesivaldo Santos and Bruno Silva Andrade. Virtual screening and molecular docking for arylalkylamine-N-Acetyltransferase (aaNAT) Inhibitors, a key enzyme of Aedes (Stegomyia) aegypti (L.) metabolism. Computational Molecular Bioscience 2015;5(3): 35-44.
4.Wright TRF. The Genetics of Biogenic-Amine Metabolism, Sclerotization, and Melanization in Drosophila melanogaster. Advances in Genetics Incorporating Molecular Genetic Medicine 1987;24:127-222.
5.Bortolato M, Chen K, Shih JC. Monoamine oxidase inactivation: from pathophysiology to therapeutics. Advanced Drug Delivery Reviews 2008;60(13-14):1527-33.
6.Wright TR. The genetics of biogenic amine metabolism, sclerotization, and melanization in Drosophila melanogaster. Advances in Genetics 1987;24:127-222.
7.Brodbeck D, Amherd R, Callaerts P, Hintermann E, Meyer UA and Affolter M. Molecular and biochemical characterization of the aaNATl (Dat) locus in Drosophila melanogaster: differential expression of two gene products. DNA and Cell Biology 2009;17(7): 621-633.
8.Amherd R, Hintermann E, Walz D, Affolter M, Meyer UA. Purification, cloning, and characterization of a second arylalkylamine N-acetyltransferase from Drosophila melanogaster. DNA and Cell Biology 2000;19(11):697-705.
9.Hardeland R, Pandi-Perumal SR, Cardinali DP. Melatonin. International Journal of Biochemistry and Cell Biology 2006;38(3):313-6.
10.Du-Xian T, Manchester LC, Reiter RJ and Calvo JR. Significance of melatonin in antioxidative defense system: reactions and products. Biological signals and receptors 2000;9(3-4):137-59.
11.Hiragaki S, Suzuki T, Mohamed AA, Takeda M. Structures and functions of insect arylalkylamine N-acetyltransferase (iaaNAT); a key enzyme for physiological and behavioral switch in arthropods. Frontiers in Physiology 2015;6:113.
12.Mayer I, Bornestaf C, Borg B. Melatonin in non-mammalian vertebrates: physiological role in reproduction? Comparative Biochemistry and Physiology Part A: Physiology 1997;118(3):515-531.
13.Coto-Montes A, Hardeland R. Antioxidative effects of melatonin in Drosophila melanogaster: Antagonization of damage induced by the inhibition of catalase. Journal of Pineal Research 1999;27(3):154-158.
14.Dyda F, Klein DC and Hickman AB. GCN5-related N-acetyltransferases: a structural overview. Annual Review of Biophysics and Biomolecular Structure 2000;29:81-103.
15.De Angelis J, Gastel J, Klein DC, Cole PA. Kinetic analysis of the catalytic mechanism of serotonin N-acetyltransferase (EC 2.3.1.87). Journal of Biological Chemistry 1998;273(5):3045-3050.
16.Tanner KG, Langer MR, Kim Y and Denu JM. Kinetic mechanism of the histone acetyltransferase GCN5 from yeast. Journal of Biological Chemistry 2000;275(29):22048-22055.
17.Holm L and Rosenström P. Dali server: conservation mapping in 3D. Nucleic Acids Research 2010; 38, W545-549.
18.Vetting MW, S de Carvalho LP, Yu M, Hegde SS, Magnet S, Roderick SL and Blanchard JS. Structure and functions of the GNAT superfamily of acetyltransferase. Archives of Biochemistry and Biophysics 2005;433(1):212-26.
19.Cheng KC, Liao JN and Lyu PC. Crystal structure of the dopamine N-acetyltransferase-acetyl-CoA complex provides insights into the catalytic mechanism. Biochemical Journal 2012;446:395-404.
20.International Union of Pure and Applied Chemistry (IUPAC) Compendium of Chemical Terminology in Gold Book second edition 1997; ISBN 0-86542-684-8.
21.Fasman, GD. Circular dichroism and the conformational analysis of biomolecules. Springer, US; 1996.
22.O’Brien R, Ladbury JE and Chowdry BZ. Isothermal titration calorimetry of biomolecules. Chapter 10 in Protein-Ligand interactions: hydrodynamics and calorimetry. Oxford University Press 2000; ISBN 0-19-963746-6.
23.Michael MP, Raman CS and Barry TN. Isothermal Titration Calorimetry of Protein–Protein Interactions. Methods 1999;19(2): 213–221.
24.Dempsey DR, Jeffries KA, Bond JD, Carpenter AM, Rodriguez-Ospina S, Breydo L, Caswell KK and Merkler DJ. Mechanistic and structural analysis of Drosophila melanogaster arylalkylamine N-acetyltransferases. Biochemistry 2014;53(49):7777-7793.
25.Otwinowski Z and Minor W. Processing of X-ray diffraction data collected in oscillation mode. Macromolecular Crystallography Part A 1997;276:307-326.
26.Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW and et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D Biological Crystallography 2010;66(Pt 2):213-21.
27.Emsley P, Lohkamp B, Scott WG and Cowtan K. Features and development of Coot. Acta Crystallographica Section D Biological Crystallography 2010;66(Pt 4):486-501.
28.DeLano, W.L. The PyMOL Molecular Graphics System (2002) on World Wide Web http://www.pymol.org
29.Laskowski RA, Macarthur MW, Moss DS and Thornton JM. Procheck - a Program to Check the Stereochemical Quality of Protein Structures. Journal of Applied Crystallography 1993;26:283-291.
30.Laskowski R A, MacArthur M W, Thornton J M (2001). PROCHECK: validation of protein structure coordinates, in International Tables of Crystallography, Volume F. Crystallography of Biological Macromolecules, eds. Rossmann M G & Arnold E, Dordrecht, Kluwer Academic Publishers, The Netherlands, pp. 722-725.
31.Eva Muñoz and Juan Sabin. Case Study: Global Analysis in ITC Displacement Titrations with AFFINImeter. Software for Science Developments.
32.Ulusu NN. Evolution of enzyme kinetic mechanisms. Journal of Molecular Evolution 2015;80(5-6):251-257.
33.Majorek KA, Kuhn ML, Chruszcz M, Anderson WF and Minor W. Structural, functional, and inhibition studies of a Gcn5-related N-acetyltransferase (GNAT) superfamily protein PA4794: a new c-terminal lysine protein acetyltransferase from Pseudomonas aeruginosa. Journal of Biological Chemistry 2013;288(42):30223-30235.
34.Dorfmueller HC, Fang WX, Rao FV, Blair DE, Attrill H and van Aalten DMF. Structural and biochemical characterization of a trapped coenzyme A adduct of Caenorhabditis elegans glucosamine-6-phosphate N-acetyltransferase 1. Acta Crystallographica Section D-Biological Crystallography 2012;68:1019-1029.
35.Peneff C, Mengin-Lecreulx D and Bourne Y. The crystal structures of apo and complexed Saccharomyces cerevisiae GNA1 shed light on the catalytic mechanism of an amino-sugar N-acetyltransferase. Journal of Biological Chemistry 2001;276(19):16328-16334.
36.Wu, H., Min, J., Zeng, H., Loppnau, P., Weigelt, J., Sundstrom, M., Arrowsmith, C.H., Edwards, A.M., Bochkarev, A. and Plotnikov, AN. Crystal structure of glucosamine-phosphate N-acetyltransferase 1.
37.Hsiao YS, Jogl G and Tong L. Crystal structures of murine carnitine acetyltransferase in ternary complexes with its substrates. Journal of Biological Chemistry 2006;281(38):28480-28487.
38.Liszczak G, Arnesen T and Marmorstein R. Structure of a ternary Naa50p (NAT5/SAN) N-terminal acetyltransferase complex reveals the molecular basis for substrate-specific acetylation. Journal of Biological Chemistry 2011;286(42):37002-37010.
39.Rojas JR, Trievel RC, Zhou JX, Mo Y, Li XM, Berger SL, Allis CD and Marmorstein R. Structure of Tetrahymena GCN5 bound to coenzyme A and a histone H3 peptide. Nature 1999;401(6748):93-98.
40.Hurtado-Guerrero R, Raimi O, Shepherd S and van Aalten DM. Glucose-6-phosphate as a probe for the glucosamine-6-phosphate N-acetyltransferase Michaelis complex. FEBS Letter 2007;581(29):5597-600.
41.Hickman AB, Namboodiri MAA, Klein DC and Dyda F. The structural basis of ordered substrate binding by serotonin N-acetyltransferase: enzyme complex at 1.8 angstrom resolution with a bisubstrate analog. Cell 1999;97(3):361-369.
42.Lawrence CK. Fragment-based drug design: tools, practical approaches, and examples. Academic Press 2011; ISBN: 9780123812742.
43.Peters T and Evans SV. Bioactive conformation I. Springer 2007; ISBN 3540490779.
44.Rudnick DA, Mcwherter CA, Rocque WJ, Lennon PJ, Getman DP and Gordon JI. Kinetic and structural evidence for a sequential ordered bi bi mechanism of catalysis by Saccharomyces cerevisiae myristoyl-coa - protein N-myristoyltransferase. Journal of Biological Chemistry 1991;266(15):9732-9739.
45.Vetting MW, de Carvalho LP, Roderick SL and Blanchard JS. A novel dimeric structure of the RimL N-acetyltransferase from Salmonella typhimurium. The Journal of Biological Chemistry 2005;280, 22108–22114.
46.Oikawa S and Akamatsu N. Three forms of rat liver glucosamine 6-phosphate acetylase and the changes in their levels during development. International Journal of Biochemistry 1985;17(1):73-80.