在果蠅中，透過乙醯基轉移酶 (N-acetyltransferase) 及乙醯輔酶A (acetyl-coenzyme A) 所進行的乙醯化作用 (N-acetylation) 是一個非常重要的過程，其涉及了神經傳遞物失活、角質層硬化和色素沉積。在西元2000年之前，在果蠅體內只發現了一種芳烴基烷基胺乙醯基轉移酶 (arylalkylamine N-acetyltransferase, AANAT)，它是多巴胺乙醯基轉移酶 (Dopamine N-Acetyltransferase, Dat)。在西元2000年之後，則有幾種推測與AANAT類似的酵素 (AANAT-like enzymes, AANATL) 被鑑定出來。這些AANATL酵素與Dat之間僅具有30%的序列同一性，但催化位和配體結合位上仍存在幾個保守性的殘基。AANATL酵素對胺類受質表現出廣泛的偏好，甚至非芳烴基烷基胺亦可作為受質，例如芳烴基烷基胺乙醯基轉移酶第八型 (AANATL8) 能夠特異性催化將醯基轉移至胍丁胺。為了更詳細地了解AANATL8的結構與活性關係，我們試圖得到AANATL8和乙醯輔酶A的二圓複合物晶體結構，但得到的晶體是多晶，並且無法計算其完整繞射數據。我們進一步建構了AANATL8、乙醯輔酶A和胍丁胺的三元複合物模擬結構模型，接著挑選可能參與對乙醯輔酶A結合、對受質結合和催化反應的關鍵候選殘基，將其分別設計成被丙胺酸取代的突變株，再分別利用等溫滴定量熱法 (Isothermal titration calorimetry, ITC) 和艾爾曼的試劑 (Ellman's reagent, DTNB) 仔細研究各突變株的配體結合能力和催化能力。結合ITC和DTNB測定的結果，我們試圖利用模擬的AANATL8三元結構來解釋這些關鍵殘基的結構與活性關係。按照有序的結合順序，首先，乙醯輔酶A進入AANATL8並與精胺酸138形成鹽橋來穩定位置；接著，胍丁胺從相對的入口進入AANATL8和乙醯輔酶A的複合物，並與麩胺酸34形成氫鍵、與色胺酸100之間的陽離子-π相互作用力來穩定位置。蘇胺酸167的羥基氧原子和胍丁胺的胺基活性氮原子之間距離為2.6 Å，因此我們認為蘇胺酸167的羥基陰離子可作為使受質胺基去質子化的常見鹼性觸媒，並啟動乙醯化作用。考慮到麩胺酸34和蘇胺酸167之間的距離為3.2 Å並且沒有找到距離更近的水分子，因此我們認為麩胺酸34可作為將蘇胺酸167去質子化的常見鹼性觸媒，就像Dat之中，麩胺酸47和絲胺酸182的關係一樣。絲胺酸171的位置則揭示其透過將硫醇鹽陰離子質子化來穩定產物輔酶A。

Acetyl coenzyme A (AcCoA) dependent N-acetylation by N-acetyltransferases in Drosophila is a very important process involved in the inactivation of neurotransmitters, cuticle sclerotization, and pigmentation. Before the year 2000, only one arylalkylamine N-acetyltransferase (AANAT) had been identified in Drosophila melanogaster, it is dopamine N-acetyltransferase (Dat). Several putative AANAT-like enzymes have been identified since 2000. These AANATL enzymes share only 30% sequence identity with Dat, but several conserved residues existed in catalytic site and ligands binding sites. AANATLs seem exhibit a broader preference for amine substrates, even non-arylalkylamine could be used as substrates, such as AANATL8 specifically catalyzes the acyl group transfer to agmatine (Agm). To understand more detail about the structure–activity relationship of AANATL8, we tried to determine the crystal structure of AANATL8/AcCoA binary complex, but the crystal was polycrystal and the diffraction data could not be calculated. We further built a simulated structural model of AANATL8/AcCoA/agmatine ternary complex. Critical residue candidates participating in AcCoA binding, substrate binding, and catalysis were designed as alanine-substituted mutants, and their ligands binding (by ITC) and catalytic ability (by DTNB assay) were carefully investigated. Combined with the results of ITC and DTNB assay, we tried to explain the structure–activity-relationship of these critical residues based on the simulated AANATL8 ternary structure. Following the sequential binding order, first, AcCoA enters AANATL8 and is stabilized by R138 via salt bridge; next, Agm enters the AANATL8-AcCoA complex from the opposite entrance and is stabilized by E34 via hydrogen bond and by W100 via a cation-π interaction. The distance between the oxygen atom in the hydroxyl group of T167 and the reactive nitrogen atom of the amine group in Agm is 2.6 Å, therefore, we propose that the hydroxylate anion of T167 can serve as a general base catalyst to deprotonate the substrate amino group and initiate the acetylation. Considering the distance between E34 and T167 is 3.2 Å and no water molecule in closer position is found, so we propose that E34 might serve as a general base to deprotonate T167, just like the relationship between E47 and S182 in Dat. The location of S171 indicates that S171 stabilizes the product CoA by protonating the thiolate anion.

中文摘要 1
Abstract 2
Acknowledgements 3
Chapter 1. Introduction 1
1.1 The ligand specificity of AgmNAT 1
1.1.1 Amine substrate 1
1.1.2 Cofactors 2
1.2 Structure of AgmNAT 2
1.3 Critical residues of AgmNAT 3
1.4 Proposed mechanism of AgmNAT 4
1.5 Aims of this study 4
1.5.1 Identification of the substrate preference 5
1.5.2 Identification of the ligands binding order 5
1.5.3 Investigation of the structure–activity relationship 5
1.5.4 Proposal for catalytic mechanism 6
Figures of Chapter 1 7
Figure 1.1 Sequence alignment of AANATLs and truncated Dat 7
Figure 1.2 The structural alignment of insect AANATs and AANATLs having known structures 8
Figure 1.3 The proposed catalytic mechanisms of AgmNAT and Dat 9
Figure 1.3 The proposed catalytic mechanisms of AgmNAT and Dat 10
Figure 1.4 Chemical structures of the tested substrates for AANATL8 11
Chapter 2. Materials and Methods 12
2.1. Materials 12
2.2. Construction of plasmid for expression of AANATL8 13
2.3. Construction of plasmids for AANATL8 mutants by site directed mutagenesis 13
2.4. Expression and purification of AANATL8 and its mutants 14
2.5. Identification of proteins 16
2.5.1. Tris-glycine SDS-PAGE 16
2.5.2. Quantification of protein concentration 16
2.5.3. Mass spectrometry 17
2.5.4. Circular dichroism spectrometry 17
2.6. DTNB assay for enzyme activity 18
2.7. Isothermal titration calorimetry 18
2.8. Bioinformatic Analysis 19
2.8.1. Molecular Docking 19
2.8.2. Pymol 19
2.8.3. Ligplot 20
2.9. Crystal condition screening 20
Tables and Figures of Chapter 2 22
Table 2.1 Oligonucleotides primers for constructing mutants of AANATL8 22
Table 2.2 Theoretical molecular weights and extinction coefficient of AANATL8 and its mutants 23
Figure 2.1 The plasmid map and sequence 24
Figure 2.2 The flow charge of protein expression and purification 25
Figure 2.3 DTNB assay 26
Chapter 3. Results and Discussion 27
3.1 Construction of pET-28a-AANATL8 27
3.2 Overexpression and purification of AANATL8 27
3.3 Enzyme specificity and catalytic abilities of AANATL8 28
3.4 Try to determine the crystal structure of AANATL8-AcCoA complex 29
3.5 Investigation into critical residues in ligands binding and catalysis by bioinformatics 30
3.6 Manufacture of AANATL8 mutants 31
3.7 The secondary structures of L8-WT and mutants 31
3.8 The enzyme activity assay of AANATL8 mutants 32
3.9 Characterization of AANATL8 mutants 33
3.10 The proposed mechanism of AANATL8 35
Tables and Figures of Chapter 3 38
Table 3.1 Molecular weight of AANATL8 38
Table 3.2 Kinetic parameters for AANATL8 and its mutants 39
Table 3.3 Ligplot+ analysis for ligands binding residues 40
Table 3.4 ITC study for AcCoA binding of AANATL8 and its mutants 41
Figure 3.1 Construction of pET-28a-AANATL8 42
Figure 3.2 Expression and purification of AANATL8 by Ni2+ column 43
Figure 3.3 Purification of tag-free AANATL8 by gel filtration 44
Figure 3.4 Identification of AANATL8 molecular weight by ESI-MS 45
Figure 3.5 Substrate specificity and catalytic ability of L8-WT based on DTNB assay 46
Figure 3.6 ITC study for the ligands binding order of L8-WT 47
Figure 3.7 Crystals and X-ray diffraction pattern of AANATL8 48
Figure 3.8 Simulated structure of AANATL8-AcCoA-agmatine ternary complex 49
Figure 3.9 Investigation into the ligand binding site of AANATL8 by Ligplot+ 50
Figure 3.10 Critical residue candidates in AANATL8 51
Figure 3.11 The results of site directed mutagenesis 52
Figure 3.12 Using protein sequence alignments to identify the mutagenesis result 53
Figure 3.13 Expression and purification of AANATL8 mutants 54
Figure 3.13 Expression and purification of AANATL8 mutants 55
Figure 3.13 Expression and purification of AANATL8 mutants 56
Figure 3.13 Expression and purification of AANATL8 mutants 57
Figure 3.14 The CD spectra of L8-WT and mutants 58
Figure 3.15 Catalytic abilities of L8-WT and its mutants based on DTNB assay 59
Figure 3.16 The Lineweaver-Burk plots of L8-WT and mutants from DTNB assay results 60
Figure 3.17 Comparison of the ITC data of L8-WT and mutants of catalytic residue candidates 61
Figure 3.18 Comparison of the ITC data of L8-WT and mutants W100A and R138A 62
Figure 3.19 Comparison of the ITC data of L8-WT and mutants E33A, F52A, and H206A 63
Figure 3.20 The roles of critical residues based on the simulated AANATL8 ternary structure 64
Figure 3.21 The steps in catalytic process of AANATL8 65
Supplementary Figure 1 The intrinsic fluorescence of W100 in AANATL8 during AcCoA titration 66
Reference 67

1. Brodbeck, D. et al. Molecular and biochemical characterization of the aaNAT1(Dat) locus in Drosophila melanogaster: differential expression of two gene products. DNA Cell Biol 17, 621-33 (1998).
2. Sloley, B.D. Metabolism of monoamines in invertebrates: the relative importance of monoamine oxidase in different phyla. Neurotoxicology 25, 175-83 (2004).
3. Andersen, S.O. Insect cuticular sclerotization: A review. Insect Biochemistry and Molecular Biology 40, 166-178 (2010).
4. Kramer, K.J. et al. Oxidative conjugation of catechols with proteins in insect skeletal systems. Tetrahedron 57, 385-392 (2001).
5. Osanai-Futahashi, M. et al. A visible dominant marker for insect transgenesis. Nature Communications 3(2012).
6. Brodbeck, D., et al. Molecular and Biochemical Characterization of the aaNATl (Dat) Locus in Drosophila melanogaster Differential Expression of Two Gene Products. DNA and cell biology 17, 621-633 (1998).
7. Dempsey, D.R. et al. Identification of an arylalkylamine N‐acyltransferase from Drosophila melanogaster that catalyzes the formation of long‐chain N‐acylserotonins. FEBS letters 588, 594-599 (2014).
8. Farrell, E.K. & Merkler, D.J. Biosynthesis, degradation and pharmacological importance of the fatty acid amides. Drug discovery today 13, 558-568 (2008).
9. Dempsey, D.R. et al. Mechanistic and structural analysis of a Drosophila melanogaster enzyme, arylalkylamine N-acetyltransferase like 7, an enzyme that catalyzes the formation of N-acetylarylalkylamides and N-acetylhistamine. Biochemistry 54, 2644-2658 (2015).
10. Dempsey, D.R., Carpenter, A.-M., Ospina, S.R. & Merkler, D.J. Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2. Insect biochemistry and molecular biology 66, 1-12 (2015).
11. Amherd, R., Hintermann, E., Walz, D., Affolter, M. & Meyer, U.A. Purification, cloning, and characterization of a second arylalkylamine N-acetyltransferase from Drosophila melanogaster. DNA and Cell Biology 19, 697-705 (2000).
12. Dempsey, D.R. et al. Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine. Sci Rep 7, 13432 (2017).
13. Dempsey, D.R. et al. Mechanistic and structural analysis of Drosophila melanogaster arylalkylamine N-acetyltransferases. Biochemistry 53, 7777-7793 (2014).
14. Kossel, A. Über das Agmatin. Hoppe-Seyler s Zeitschrift für physiologische Chemie 66, 257-261 (1910).
15. Piletz, J.E. et al. Agmatine: clinical applications after 100 years in translation. Drug discovery today 18, 880-893 (2013).
16. Galea, E., Regunathan, S., Eliopoulos, V. & FEINSTEIN, D.L. Inhibition of mammalian nitric oxide synthases by agmatine, an endogenous polyamine formed by decarboxylation of arginine. Biochemical Journal 316, 247-249 (1996).
17. Gadkari, T.V., Cortes, N., Madrasi, K., Tsoukias, N.M. & Joshi, M.S. Agmatine induced NO dependent rat mesenteric artery relaxation and its impairment in salt-sensitive hypertension. Nitric Oxide 35, 65-71 (2013).
18. Marc, R.E. Mapping glutamatergic drive in the vertebrate retina with a channel‐permeant organic cation. Journal of Comparative Neurology 407, 47-64 (1999).
19. Mistry, S.K. et al. Cloning of human agmatinase. An alternate path for polyamine synthesis induced in liver by hepatitis B virus. American Journal of Physiology-Gastrointestinal and Liver Physiology 282, G375-G381 (2002).
20. Dallmann, K. et al. Human agmatinase is diminished in the clear cell type of renal cell carcinoma. International journal of cancer 108, 342-347 (2004).
21. Morris, S.M. Vertebrate agmatinases: what role do they play in agmatine catabolism ANNALS-NEW YORK ACADEMY OF SCIENCES, 30-33 (2003).
22. Satriano, J. Arginine pathways and the inflammatory response: interregulation of nitric oxide and polyamines. Amino acids 26, 321-329 (2004).
23. Uzbay, T.I. The pharmacological importance of agmatine in the brain. Neuroscience & Biobehavioral Reviews 36, 502-519 (2012).
24. Moncada, S. Mechanisms of disease-the l-arginine nitric-oxide pathway. N Engl J Med 329, 2002-2012 (1993).
25. Moinard, C., Cynober, L. & de Bandt, J.-P. Polyamines: metabolism and implications in human diseases. Clinical nutrition 24, 184-197 (2005).
26. Hickman, A.B., Klein, D.C. & Dyda, F. Melatonin biosynthesis: the structure of serotonin N-acetyltransferase at 2.5 A resolution suggests a catalytic mechanism. Mol Cell 3, 23-32 (1999).
27. Hickman, A.B., Namboodiri, M.A.A., Klein, D.C. & 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 97, 361-369 (1999).
28. Cheng, K.C., Liao, J.N. & Lyu, P.C. Crystal structure of the dopamine N-acetyltransferase-acetyl-CoA complex provides insights into the catalytic mechanism. Biochem J 446, 395-404 (2012).
29. Han, Q., Robinson, H., Ding, H.Z., Christensen, B.M. & Li, J.Y. Evolution of insect arylalkylamine N-acetyltransferases: Structural evidence from the yellow fever mosquito, Aedes aegypti. Proceedings of the National Academy of Sciences of the United States of America 109, 11669-11674 (2012).
30. Vetting, M.W. et al. Structure and functions of the GNAT superfamily of acetyltransferases. Arch Biochem Biophys 433, 212-26 (2005).
31. Dyda, F., Klein, D.C. & Hickman, A.B. GCN5-related N-acetyltransferases: A structural overview. Annual Review of Biophysics and Biomolecular Structure 29, 81-103 (2000).
32. Salah Ud-Din, A.I., Tikhomirova, A. & Roujeinikova, A. Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT). Int J Mol Sci 17(2016).
33. Vetting, M.W. et al. Structure and functions of the GNAT superfamily of acetyltransferases. Archives of biochemistry and biophysics 433, 212-226 (2005).
34. Wolf, E. et al. Crystal structure of a GCN5-related N-acetyltransferase: Serratia marcescens aminoglycoside 3-N-acetyltransferase. Cell 94, 439-449 (1998).
35. Pavlicek, J. et al. Evidence that proline focuses movement of the floppy loop of arylalkylamine N-acetyltransferase (EC 2.3. 1.87). Journal of Biological Chemistry 283, 14552-14558 (2008).
36. Ellman, G.L. Tissue sulfhydryl groups. Archives of biochemistry and biophysics 82, 70-77 (1959).
37. Collier, H.B. A note on the molar absorptivity of reduced Ellman's reagent, 3-carboxylato-4-nitrothiophenolate. Analytical biochemistry 56, 310 (1973).
38. Riddles, P.W., Blakeley, R.L. & Zerner, B. Reassessment of Ellman's reagent. in Methods in enzymology, Vol. 91 49-60 (Elsevier, 1983).
39. Riener, C.K., Kada, G. & Gruber, H.J. Quick measurement of protein sulfhydryls with Ellman's reagent and with 4, 4′-dithiodipyridine. Analytical and bioanalytical chemistry 373, 266-276 (2002).
40. Wiseman, T., Williston, S., Brandts, J.F. & Lin, L.-N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Analytical biochemistry 179, 131-137 (1989).
41. Saponaro, A. Isothermal Titration Calorimetry: A Biophysical Method to Characterize the Interaction between Label-free Biomolecules in Solution. Bio-protocol 8(2018).
42. Duhovny, D., Nussinov, R. & Wolfson, H.J. Efficient unbound docking of rigid molecules. in International workshop on algorithms in bioinformatics 185-200 (Springer, 2002).
43. Schneidman-Duhovny, D., Inbar, Y., Nussinov, R. & Wolfson, H.J. PatchDock and SymmDock: servers for rigid and symmetric docking. Nucleic acids research 33, W363-W367 (2005).
44. Wallace, A.C., Laskowski, R.A. & Thornton, J.M. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein engineering, design and selection 8, 127-134 (1995).
45. Laskowski, R.A. & Swindells, M.B. LigPlot+: multiple ligand–protein interaction diagrams for drug discovery. (ACS Publications, 2011).
46. Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 1.8. (2015).
47. Manavalan, P. & Johnson Jr, W.C. Variable selection method improves the prediction of protein secondary structure from circular dichroism spectra. Analytical biochemistry 167, 76-85 (1987).
48. Rodger, A. & Nordén, B. Circular dichroism and linear dichroism, (Oxford University Press, USA, 1997).
49. Zhong, W. et al. From ab initio quantum mechanics to molecular neurobiology: a cation-pi binding site in the nicotinic receptor. Proc Natl Acad Sci U S A 95, 12088-93 (1998).