胍丁胺乙醯基轉移酶 (AgmNAT) 專門催化乙醯基從乙醯輔酶A轉移到胍丁胺上，並且屬於芳烴基烷基胺乙醯基轉移酶家族。這類的乙醯化作用會影響許多生物功能，例如神經傳遞物質的失活、睡眠覺醒週期、色素沉澱和角質層硬化。這是第一個關於胍丁胺乙醯基轉移酶二元體和三元體的研究。我們解出了胍丁胺乙醯基轉移酶突變蛋白(丙胺酸171突變株)的單元體及二元體/三元體的晶體結構並且進一步去比較他們的差異。在單元體和二元體/三元體之間可觀察到明顯的構型變化，而在二元體和三元體之間未發現到顯著差異。最有趣的構型變化是在loopL137-N141移向乙醯輔酶A的入口，並導致麩胺酸33和精胺酸138之間會形成鹽橋以穩定乙醯輔酶A的結合。根據胍丁胺乙醯基轉移酶突變蛋白(丙胺酸171突變株)三元體的結構，選擇了幾個殘基設計成被丙胺酸取代的突變株以測試其作用。最後，我們提出精胺酸138和酪胺酸170在乙醯輔酶A的結合中為重要的殘基，而天門冬胺酸49直接參與穩定胍丁胺的結合，再結合胍丁胺乙醯基轉移酶突變蛋白(丙胺酸171突變株)的結構和功能測試結果進一步提出了催化機制。

Agmatine N-acetyltransferase (AgmNAT) specifically catalyzes the transfer of acetyl group from acetyl coenzyme A (Ac-CoA) to agmatine (Agm) and belongs to arylalkylamine N-acetyltransferase (AANAT) family. This Ac-CoA-dependent N-acetylation has impact on many bio-functions such as the inactivation of neurotransmitters, sleep-wake cycle, pigmentation, and cuticle sclerotization. This study is the first report of the binary and ternary complexes of AgmNAT. We have determined the crystals of apo form and binary/ternary complexes of AgmNAT-S171A and further compared them. Recognizable conformational changes were observed between apo form and complex forms while no significant difference was detected between binary and ternary complexes. The most interesting conformational change were a loopL137-N141 shifting toward the entrance of Ac-CoA in complex forms and resulted in the formation of a salt bridge between E33 and R138 to stabilize Ac-CoA binding. According to the structure of ternary complex of AgmNAT-S171A, several residues were selected for alanine-scanning mutagenesis to test their roles. Finally, we proposed that R138 and Y170 play important roles in Ac-CoA binding and D49 participates in stabilizing Agm binding directly. Combined with the structures of AgmNAT-S171A complex forms and the results of functional test, we further proposed the catalytic mechanism.

1. O'Flynn, B.G., Suarez, G., Hawley, A.J. & Merkler, D.J. Insect Arylalkylamine N-Acyltransferases: Mechanism and Role in Fatty Acid Amide Biosynthesis. Frontiers in molecular biosciences 5(2018).
2. Dempsey, D.R. Identification and Characterization of N-acyltransferase Enzymes that are Involved in the Biosynthesis of Fatty Acid Amides. (2015).
3. 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).
4. Dempsey, D.R. et al. Mechanistic and structural analysis of Drosophila melanogaster arylalkylamine N-acetyltransferases. Biochemistry 53, 7777-7793 (2014).
5. Ganguly, S., Coon, S.L. & Klein, D.C. Control of melatonin synthesis in the mammalian pineal gland: the critical role of serotonin acetylation. Cell and tissue research 309, 127-137 (2002).
6. Hardeland, R. & Poeggeler, B. Non‐vertebrate melatonin. Journal of pineal research 34, 233-241 (2003).
7. SCHOMERUS, C. & KORF, H.W. Mechanisms regulating melatonin synthesis in the mammalian pineal organ. Annals of the New York Academy of Sciences 1057, 372-383 (2005).
8. Reiter, R.J. The pineal gland and melatonin in relation to aging: a summary of the theories and of the data. Experimental gerontology 30, 199-212 (1995).
9. Hickman, A.B., Klein, D.C. & Dyda, F. Melatonin biosynthesis: the structure of serotonin N-acetyltransferase at 2.5 Å resolution suggests a catalytic mechanism. Molecular cell 3, 23-32 (1999).
10. Klein, D.C. Arylalkylamine N-acetyltransferase:“the Timezyme”. Journal of biological chemistry 282, 4233-4237 (2007).
11. Osanai-Futahashi, M. et al. A visible dominant marker for insect transgenesis. Nature communications 3, 1295 (2012).
12. 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).
13. Sloley, B.D. Metabolism of monoamines in invertebrates: the relative importance of monoamine oxidase in different phyla. Neurotoxicology 25, 175-183 (2004).
14. Kramer, K.J. et al. Oxidative conjugation of catechols with proteins in insect skeletal systems. Tetrahedron 57, 385-392 (2001).
15. Andersen, S.O. Insect cuticular sclerotization: a review. Insect biochemistry and molecular biology 40, 166-178 (2010).
16. Wright, T.R. The genetics of biogenic amine metabolism, sclerotization, and melanization in Drosophila melanogaster. in Advances in genetics, Vol. 24 127-222 (Elsevier, 1987).
17. 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).
18. Farrell, E.K. & Merkler, D.J. Biosynthesis, degradation and pharmacological importance of the fatty acid amides. Drug discovery today 13, 558-568 (2008).
19. 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).
20. 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).
21. 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).
22. 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).
23. Li, H.-J. National Tsing Hua University (2019).
24. 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).
25. Vetting, M.W. et al. Structure and functions of the GNAT superfamily of acetyltransferases. Archives of biochemistry and biophysics 433, 212-226 (2005).
26. 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).
27. Yang, Y.C. National Tsing Hua University (2016).
28. Ding, W.C. National Tsing Hua University (2016).
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. 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. Biochemical Journal 446, 395-404 (2012).
31. Salah Ud-Din, A.I.M., Tikhomirova, A. & Roujeinikova, A. Structure and functional diversity of GCN5-related N-acetyltransferases (GNAT). International journal of molecular sciences 17, 1018 (2016).
32. 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).
33. Wolf, E. et al. Crystal structure of a GCN5-related N-acetyltransferase: Serratia marcescens aminoglycoside 3-N-acetyltransferase. Cell 94, 439-449 (1998).
34. Singh, S. et al. Monoamine-and histamine-synthesizing enzymes and neurotransmitters within neurons of adult human cardiac ganglia. Circulation 99, 411-419 (1999).
35. Kurian, M.A., Gissen, P., Smith, M., Heales, S.J. & Clayton, P.T. The monoamine neurotransmitter disorders: an expanding range of neurological syndromes. The Lancet Neurology 10, 721-733 (2011).
36. Evans, P.D. Biogenic amines in the insect nervous system. in Advances in insect physiology, Vol. 15 317-473 (Elsevier, 1980).
37. Monastirioti, M. Biogenic amine systems in the fruit fly Drosophila melanogaster. Microscopy research and technique 45, 106-121 (1999).
38. Shalaby, A.R. Significance of biogenic amines to food safety and human health. Food research international 29, 675-690 (1996).
39. Marc, R.E. Mapping glutamatergic drive in the vertebrate retina with a channel‐permeant organic cation. Journal of Comparative Neurology 407, 47-64 (1999).
40. Halaris, A. & Plietz, J. Agmatine. CNS drugs 21, 885-900 (2007).
41. Piletz, J.E. et al. Agmatine: clinical applications after 100 years in translation. Drug discovery today 18, 880-893 (2013).
42. Uzbay, T.I. The pharmacological importance of agmatine in the brain. Neuroscience & Biobehavioral Reviews 36, 502-519 (2012).
43. Galea, E., Regunathan, S., Eliopoulos, V., FEINSTEIN, D.L. & REIS, D.J. Inhibition of mammalian nitric oxide synthases by agmatine, an endogenous polyamine formed by decarboxylation of arginine. Biochemical Journal 316, 247-249 (1996).
44. 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).
45. Moncado, S. & Higgs, A. Mechanisms of disease–the L-arginine nitric oxide pathway. N Eng J Med 329(1993).
46. 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).
47. 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).
48. Morris, S.M. Vertebrate agmatinases: what role do they play in agmatine catabolism? ANNALS-NEW YORK ACADEMY OF SCIENCES, 30-33 (2003).
49. Satriano, J. Arginine pathways and the inflammatory response: interregulation of nitric oxide and polyamines. Amino acids 26, 321-329 (2004).
50. Moinard, C., Cynober, L. & de Bandt, J.-P. Polyamines: metabolism and implications in human diseases. Clinical Nutrition 24, 184-197 (2005).
51. Coleman, C.S., Hu, G. & Pegg, A.E. Putrescine biosynthesis in mammalian tissues. Biochemical Journal 379, 849-855 (2004).
52. Moncada, S. Mechanisms of disease-the l-arginine nitric-oxide pathway. N Engl J Med 329, 2002-2012 (1993).
53. Dempsey, D.R. et al. Structural and mechanistic analysis of Drosophila melanogaster agmatine N-acetyltransferase, an enzyme that catalyzes the formation of N-acetylagmatine. Scientific reports 7, 13432 (2017).
54. 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).
55. Powell, H.R. X-ray data processing. Bioscience reports 37, BSR20170227 (2017).
56. Otwinowski, Z., Minor, W., Borek, D. & Cymborowski, M. Denzo and scalepack. International Tables for Crystallography, 282-295 (2006).
57. Otwinowski, Z. Minor W 1997 Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276, 307326.
58. DeLano, W. The PyMOL Molecular Graphics System. DeLano Scientific; San Carlos, CA, USA: 2002. (2002).
59. 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).
60. Saponaro, A. Isothermal Titration Calorimetry: A Biophysical Method to Characterize the Interaction between Label-free Biomolecules in Solution. Bio-protocol 8(2018).
61. Ellman, G.L. Tissue sulfhydryl groups. Archives of biochemistry and biophysics 82, 70-77 (1959).
62. Collier, H.B. A note on the molar absorptivity of reduced Ellman's reagent, 3-carboxylato-4-nitrothiophenolate. Analytical biochemistry 56, 310 (1973).
63. Riddles, P.W., Blakeley, R.L. & Zerner, B. Reassessment of Ellman's reagent. in Methods in enzymology, Vol. 91 49-60 (Elsevier, 1983).
64. 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).
65. Lineweaver, H. & Burk, D. The determination of enzyme dissociation constants. Journal of the American chemical society 56, 658-666 (1934).
66. Johnson, K.A. & Goody, R.S. The original Michaelis constant: translation of the 1913 Michaelis–Menten paper. Biochemistry 50, 8264-8269 (2011).
67. 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).
68. Rodger, A. & Nordén, B. Circular dichroism and linear dichroism, (Oxford University Press, USA, 1997).