鹽橋在芳烷基胺乙醯基轉移酶之結構與功能探討

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研究生：	許育碩 Hsu, Yu-Shuo
論文名稱：	鹽橋在芳烷基胺乙醯基轉移酶之結構與功能探討 Structural and functional insights into the role of the salt bridge in arylalkylamine N-acetyltransferase
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:	鄭惠春 Cheng, Hui-Chun 吳昆峯 Wu, Kuen-Phon
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	80
中文關鍵詞：	乙醯基轉移酶、乙醯輔酶A 、X射線晶體學、芳烷基胺乙醯轉移酶、胍丁胺、等溫滴定量熱法
外文關鍵詞：	N-acetyltransferase, acetyl-coenzymeA, X-ray crystallography, arylalkylamine N-acetyltransferase, agmatine, ITC
相關次數：	點閱：3 下載：0
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芳烷基胺乙醯基轉移酶(AANAT)負責將乙醯基從乙醯輔酶A(AcCoA)轉移到芳烷基胺上，並且參與許多生物體的生理功能。芳烷基胺乙醯基轉移酶的催化循環遵循有順序的機制，需先與輔因子(AcCoA)結合，再與基質結合，之後完成乙醯基轉移反應。其催化循環可以分為四個階段，包括初始階段、輔因子結合階段、基質結合階段和反應階段。其中在初始階段和輔因子結合階段之間可觀察到顯著的結構變化，最顯著的變化是P環(P-loop)向乙醯輔酶A的入口移動，同時P環上的精胺酸和在α1上的麩胺酸/天冬胺酸會形成鹽橋以穩定這個P環移動。此外，基質結合口袋和催化位點也在這個階段形成。在這項研究中，我們在兩種芳烷基胺乙醯基轉移酶中建構了鹽橋突變體，分別是多巴胺乙醯基轉移酶(Dat)和胍丁胺乙醯基轉移酶(AgmNAT)。透過DTNB測定酵素活性、使用等溫滴定微量熱儀(ITC)量測配體結合親和力，並利用晶體繞射解析他們的結構變化。在我們的研究中顯示缺少鹽橋會大幅降低配體結合能力和酶活性，但在胍丁胺乙醯基轉移酶突變體中基質結合能力並未降低。從多巴胺乙醯基轉移酶突變體 Dat-D46A的結構沒有觀察到P環移動，而且負責催化的殘基和底物結合的殘基的側鏈們都沒有移動到正確的位置。此外，也沒有形成完整的配體結合通道來調控配體的進出。胍丁胺乙醯基轉移酶突變體AgmNAT-R138A-S171A的結構揭示了與Dat-D46A相似的結果。因此，這種保守鹽橋在芳烷基胺乙醯基轉移酶的作用可能是一種結構轉換開關，由輔因子結合觸發，然後促進底物結合口袋和催化位點的形成。

Arylalkylamine N-acetyltransferase (AANAT) is responsible for acetyl transfer from acetyl coenzyme A (AcCoA) to arylalkylamine and involved in many physiological functions. The catalytic cycle of AANAT follows an ordered sequential mechanism and can be recognized as several stages, including the initial stage (apo form), cofactor binding stage, substrate binding stage, and catalysis stage. Comparison of AANATs structures in apo form and complexes reveals that significant conformational changes are observed between the initial stage and cofactor binding stage. The most significant changes are the shift of P-loop toward the bound cofactor to stabilize it, while a salt bridge newly formed by Glu/Asp on α1 and Arg on P-loop to fix this movement. The substrate binding pocket and catalytic site are also formed in this cofactor binding stage. In this study, we constructed mutants of the salt bridge in two AANATs, Dopamin N-acetyltransferase (Dat) and Agmatine N-acetyltransferase (AgmNAT), and then surveyed their acetyl transfer activities by DTNB assay, ligands binding affinities by isothermal titration calorimetry (ITC), and structures by crystallography. Our data revealed that the loss of salt bridge largely reduced the ligands binding abilities and catalytic activity, but the substrate binding abilities was not reduced in AgmNAT mutants. Structures of Dat-D46A showed that the P-loop movement was not observed, while the side chains of catalytic residues and substrate binding residues did not move to the correct positions. In addition, whole ligands binding tunnel was not formed well to control the entry and exit of ligands. The AgmNAT-R138A-S171A structures revealed the similar results to that from Dat-D46A. Therefore, the role of this conserved salt bridge may be a structural switch, which is triggered by cofactor binding, then facilitates the formation of the substrate binding pocket and catalytic site.

Chapter 1. Introduction    1
1.1. Overview of the General control non-repressible 5 (GCN5) related
N-acetyltransferase (GNAT) superfamily    1
1.2. Arylalkylamine N-acetyltransferase (AANAT)    2
1.2.1. The functions of AANATs    2
1.2.2. The structures of AANATs    3
1.2.3. The chemical mechanism and catalytic cycle of AANATs    4
1.2.4. AgmNAT    5
1.3. Conformational change during catalytic cycle    6
1.4. Aim of this study    7
Tables and Figures of Chapter 1    8
Figure 1.1. Topology of GNATs superfamily    8
Figure 1.2. The complex structure of Dat    9
Figure 1.3. Comparison of AANATs with known structures    10
Figure 1.4. The proposed catalytic cycle of Dat    11
Figure 1.5. Comparison of substrate entrance of Dat and AgmNAT    12
Figure 1.6. Comparison of AANAT in different stages of catalytic cycle    13
Figure 1.7. Detail changes of catalytic site in DmDat    14
Figure 1.8. Detail changes of substrate binding tunnel in DmDat    15
Figure 1.9. No large changes of critical residues in DmAgmNAT    16
Figure 1.10. The salt bridge in AANATs of different species    17
Chapter 2. Materials and Methods    18
2.1. Materials    18
2.2. Production of recombinant AgmNAT and its mutants    19
2.2.1. Construction of the expression plasmids    19
2.2.2. Expression and purification    20
2.3. Production and purification of Dat and its mutants    21
2.4. Quantification of protein concentration    24
2.5. Protein electrophoresis    24
2.6. Circular dichroism (CD)    25
2.7. Acetyltransferase activity assay    25
2.8. Isothermal titration calorimetry (ITC).    26
2.8. Crystallization and structure determination    27
2.8.1. AgmNAT-WT and its mutants    27
2.8.2. The salt bridge mutants of Dat    28
2.8.3. Data collection and process    28
Tables and Figures of Chapter 2    30
Table 2.1. Oligonucleotides primers for constructing mutants of AgmNAT    30
Table 2.2. The mutagenesis reaction compositions and
thermocycling conditions    31
Table 2.3. Theoretical molecular weights and extinction coefficient of
AgmNAT and its mutants.    32
Table 2.4. The reaction compositions of DTNB assay for
AgmNAT and its mutants.    33
Table 2.5. The reaction compositions of DTNB assay for
Dat and its mutants    34
Figure 2.1. Flow chart of AgmNAT expression and purification    35
Figure 2.2. Flow chart of Dat expression and purification    36
Chapter 3. Results and Discussion    37
3.1. Construction and production of mutants of the conserved salt bridge    37
3.2. The enzyme activity    37
3.2.1. The enzyme activity of Dat and its mutants    37
3.3.2. The enzyme activity of AgmNAT and its mutants    38
3.2.3. The role of the conservatively formed salt bridge in
catalyzing N-acetylation    38
3.3. Cofactor binding ability and substrate binding ability    39
3.3.1. The binding ability of Dat and its mutants    39
3.3.2. The binding ability of AgmNAT and its mutants    40
3.3.3. The role of the conservatively formed salt bridge in
cofactor binding and substrate binding    41
3.4. Structure-based Analyses    42
3.4.1. Structural analyses of Dat-D46A    42
3.4.2. Structural analyses of AgmNAT-R138A-S171A    44
3.4.3. Structural analyses of AgmNAT-WT complex    46
3.5. Comparison of Dat, AgmNAT, and their mutants    47
Tables and Figures of Chapter 3    50
Table 3.1. Kinetic parameters for Dat, AgmNAT and their mutants    50
Table 3.2. ITC study for ligands binding of Dat, AgmNAT and their mutants    51
Table 3.3. Crystallization condition of Dat and its mutants    52
Table 3.4. Crystallization condition of AgmNAT and its mutants    53
Table 3.5. Data collection and refinement statistics    54
Figure 3.1. The CD spectra of Dat, AgmNAT and their mutants    55
Figure 3.2. Enzyme activity of Dat, AgmNAT and their mutants    56
Figure 3.3. The ligands binding tests of Dat and its mutants by ITC    57
Figure 3.4. The substrate binding tests of Dat and its mutants by ITC    58
Figure 3.5. The ligands binding tests of AgmNAT and its mutants by ITC    59
Figure 3.6. Identification of CoA only in Dat-D46A from
ternary cocrystallization    60
Figure 3.7. Structural analyses of Dat-D46A    61
Figure 3.8. The change of substrate binding tunnel in Dat proteins    62
Figure 3.9. Comparison of the structure of AgmNAT-R138A-S171A complex    63
Figure 3.10. Comparison of AgmNAT-R138A-S171A and AgmNAT-S171A    64
Figure 3.11. Comparison of the structure of AgmNAT-WT complex    65
Figure 3.12. Comparison of AgmNAT-WT and AgmNAT-S171A    66
Chapter 4. Conclusion    67
Appendix    69
Appendix I. Production of Dat, AgmNAT and their mutants    69
Appendix II. The crystallization tests    69
Appendix Figure 1. The results of site directed mutagenesis    70
Appendix Figure 2. Expression and purification results    72
Appendix Table 1. Crystallization conditions and results of
Dat, AgmNAT and their mutants    73
Reference    76

                                

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