果蠅乙醯基轉移酶類似蛋白第八型之結構暨功能分析 - 揭示其酶活性所需的關鍵殘基

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研究生：	李賀傑 Li, He-Jie
論文名稱：	果蠅乙醯基轉移酶類似蛋白第八型之結構暨功能分析 - 揭示其酶活性所需的關鍵殘基 Structure-function analyses of Drosophila melanogaster Arylalkylamine N-Acetyltransferases Like 8 protein - uncovering the key residues required for enzyme activity
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:	蘇士哲 Sue, Shih-Che 蕭乃文 Hsiao, Nai-Wan
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：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	70
中文關鍵詞：	乙醯基轉移酶、乙醯化作用、乙醯輔酶A 、芳烴基烷基胺、等溫滴定量熱法、艾爾曼的試劑
外文關鍵詞：	N-acetyltransferase, N-acetylation, acetyl-coenzymeA, arylalkylamine, ITC, DTNB
相關次數：	點閱：3 下載：0
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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

                                

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