果蠅乙醯基轉移酶類似蛋白第二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：	羅正銘 Lo, Cheng-Ming
論文名稱：	果蠅乙醯基轉移酶類似蛋白第二型之結構暨功能分析 - 揭示其酶活性所需的關鍵殘基 Structure-function analysis of Drosophila melanogaster Arylalkylamine N-Acetyltransferases Like 2 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：	108
語文別：	英文
論文頁數：	74
中文關鍵詞：	乙醯基轉移酶、乙醯化作用、乙醯輔酶A 、等溫滴定微量熱儀、結合親和力、熱力學、艾爾曼的試劑、定位突變、酵素動力學、蛋白質結構
外文關鍵詞：	Arylalkylamine N-acetyltransferase like, Acetyl-coenzyme A, binding order in radom fashion, Ellman's reagent
相關次數：	點閱：3 下載：0
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　　在果蠅中，經由乙醯輔酶A(acetyl-coenzyme A)所進行的乙醯化作用(N-acetylation)是個重要的步驟，其涉及了神經傳導物質的分解、褪黑素的合成、角質層硬化、色素沉澱及晝夜週期。現今，只有四個芳烴基烷基胺乙醯基轉移酶(arylalkylamine N-acetyltransferase, AANAT)被解析出結構，其中之一就是多巴胺乙醯基轉移酶(dopamine N-acetyltransferase, Dat)。它是在黑腹果蠅中唯一被辨認的。但2000年，有幾種推測與AANAT類似的酵素被鑑定出來。這些AANATL酵素和Dat之間有30%的序列相同性和50%的序列相似性，但是他們也催化乙醯化作用。我們對AANATL2非常有興趣，因為它以相同的芳烴基烷基胺作為受質，但能接受較長鏈的醯基輔酶作為輔因子。在蛋白質與配體結合的分析中，我們發現AANATL2可以分別結合受質與輔因子。這意味著AANATL2遵循著隨機結合順序，然而，其他AANATLs與AANATs需要有順序地結合配體。為了更詳細地瞭解AANATL2，我們試圖得到AANATL2晶體結構。但非常不幸運的，全部得到的晶體皆無法使用。為了辨別出AANATL2關鍵的殘基，我們將Dat之序列與結構對齊分析，以決定突變與功能分析的候選殘基。被挑選的殘基麩胺酸29、絲胺酸167與絲胺酸171和催化有關，麩胺醯胺28與精胺酸138和輔因子結合有關，苯丙胺酸25和受質結合有關。半胱胺酸166接近催化位且涉及維持催化位結構。首先，突變株在圓二色性(CD)光譜上分析，二級結構大致上都有形成且與野生型相似，以確認突變株皆不影響結構。結合ITC、DTNB分析與模擬結構分析的結果，我們試圖解釋這些關鍵殘基在結構與活性上的關係。所有突變株相對於野生型在催化活性上都有衰退，絲胺酸171突變株甚至完全失去催化能力。苯丙胺酸25突變株被分析顯示造成受質與乙醯輔酶A結合力下降。麩胺醯胺28突變株將造成催化活性下降且不影響乙醯輔酶A結合，所以我們認為其區域性影響麩胺酸29。精胺酸138突變株確實造成乙醯輔酶A結合力下降。半胱胺酸166具保守性且接近絲胺酸167，半胱胺酸166突變株顯示沒有影響乙醯輔酶A結合的結果，所以我們認為其區域性影響絲胺酸167，造成催化能力下降。我們的數據顯示，麩胺酸29、絲胺酸167與絲胺酸171參與了催化，如同Dat的催化三元組，然而，絲胺酸167也影響輔因子的結合。本篇研究發現了AANATL2與其它AANATs或AANATLs顯著不同之處，就是AANATL2遵循著隨機結合順序，本篇研究也提供我們對於AANATL2的特性與功能殘基更多的了解。

　　Acetyl coenzyme A (Ac-CoA) dependent N-acetylation in Drosophila is a critical process involved in neurotransmitter catabolism, biosynthesis of melatonin, cuticle sclerotization, pigmentation and circadian cycle. To date, only four arylalkylamine N-acetyltransferases (AANATs) have known structures. One of them is dopamine N-acetyltransferase (Dat), which is the only one identified AANAT in Drosophila melanogaster. But since year 2000, several putative AANAT-like enzymes have been identified in Drosophila melanogaster. The AANAT-like enzymes share only 30% sequence identity and 50% similarity with Dat, but they also catalyze the N-acetylation. We were interested in AANATL2 because it used the same arylalkylamine substrate, but accept longer chain acyl-CoA as cofactor. In protein-ligands binding assay (by isothermal titration calorimetry, ITC), we found out that AANATL2 can bind to Ac-CoA and PEA individually. It means that AANATL2 follows a binding mechanism in random fashion, however other AANATs or AANATLs follows an ordered sequential mechanism. To understand more detail about the structure-activity relationship of AANATL2, we tried to determine the crystal structures of AANATL2 apo form and AANATL2/Ac-CoA binary complex. But unfortunately, all crystals data was unusable and probably was salt. To identify the critical residues in AANATL2, alignments of sequence and structure with Dat were both used to decide the candidates for mutagenesis and functional assay. The selected residues were catalysis related E29, S167, and S171, Ac-CoA binding related Q28 and R138, as well as substrate binding related F25. C166 was close to active site and may involve in holding the structure of active site. Based on circular dichroism (CD) data, the overall secondary structures of mutants are similar to that of AANATL2 wild-type, so we confirmed that these residues did not affect structures. Combined with the results of ligands binding (ITC), catalysis function (DTNB assay), and structural analysis of modelling AANATL2 ternary complex, we tried to explain the structure-activity relationship of these selected residues. The catalytic abilities of all mutants were reduced in DTNB assay, while S171A even totally lost the catalytic capability. The ITC data revealed that F25A not only largely lost the binding ability to substrate, but also had decreased Ac-CoA binding ability. Q28A did not affect Ac-CoA binding but still resulted in loss of catalysis, so we proposed that Q28 may affect E29 via unclear local effect. R138A indeed caused significantly decreased Ac-CoA binding affinity. C166 as a highly conserved residue next to S167, C166A showed no influence to Ac-CoA binding but only kept 60% catalysis activity. Therefore, we proposed that C166A may offer local effect on S167, and resulted in the loss of catalysis. Our data indicated that E29, S167 and S171 may participate in catalysis, like their corresponding catalytic triad in Dat, while S167A also reduced Ac-CoA binding. This study found out a significant difference among AANATL2 and other AANATs or AANATLs, which is that AANATL2 binds to ligands in a random fashion. And this study also provides us a better understanding of the enzyme characteristics and functional residues of AANATL2.

Chapter 1. Introduction    1
Figures of Chapter 1    5
Figure 1.1 Topology of GNATs superfamily    5
Figure 1.2 Structures of Dat in binary and ternary complexes    6
Figure 1.3 The proposed catalytic mechanisms of Dat    7
Figure 1.4 Sequence alignment of AANATLs and truncated Dat    8
Chapter 2. Material and Methods    9
2.1 Materials    9
2.2 Construction of recombinant AANATL2 plasmid    9
2.3 Construction of recombinant AANATL2 mutant plasmids    10
2.4 Expression of AANATL2 and its mutants    11
2.5 Purification of AANATL2 and its mutants    11
2.6 SDS-PAGE analysis    12
2.7 Identification of AANATL2 based on its molecular weight    13
2.8 Crystallization and Data Collection    13
2.9 Circular dichroism spectrometry    13
2.10 Substrates specificity screening and catalytic ability test by DTNB assay    14
2.11 ITC binding assay    15
2.12 Molecular modeling and docking    15
2.13 Ligplot    16
Tables and Figures of Chapter 2    17
Table 2.1 Primers for constructing site-directed mutagenesis of AANATL2    17
Table 2.2 Theoretical molecular weights and extinction coefficients of AANATL2 and its mutants    18
Table 2.3 The screening kit of AANATL2 crystallization    19
Table 2.4 Structure of tested substrates for AANATL2    20
Table 2.5 The concentration and volume of the substrate and protein for DTNB assay    21
Figure 2.1 The plasmid maps of U5479CI180-1 and pAANATL2    22
Figure 2.2 Sequence of tag-fused AANATL2    23
Figure 2.3 The flow charge of protein expression and purification    24
Figure 2.4 DTNB assay mechanism    25
Chapter 3. Results and Discussion    26
3.1 Reconstruction of expression plasmid pAANATL2    26
3.2 Expression and Purification of AANATL2    26
3.3 Protein crystallization for x-ray diffraction    27
3.4 The substrates screening of AANATL2    28
3.5 Functional assay for AANATL2 by DTNB    28
3.6 Identification of the ligands binding order by ITC    29
3.7 Selecting the critical residue candidates by sequence alignment, structural alignment, and Ligplot+ analysis    29
3.8 Production of AANATL2 mutants    32
3.9 Secondary structures of AANATL2 and its mutants    32
3.10 The enzyme activity assay of AANATL2 and its mutants    32
3.11 Ligands binding abilities of mutants    33
3.12 Characterization of the critical residues in AANATL2    35
3.13 Conclusion    36
Tables and Figures of Chapter 3    39
Table 3.1 Ligplot+ analysis for ligands binding residues    39
Table 3.2 Kinetic parameters of AANATL2 and its mutants    40
Table 3.3 ITC studies for Ac-CoA binding of AANATL2 and its mutants    41
Table 3.4 ITC studies for PEA binding of AANATL2 and it mutants    42
Figure 3.1 The DNA gel of cutting fragment and sequencing result    43
Figure 3.2 Purification of AANATL2 by Ni2+ -affinity column    44
Figure 3.3 Identification of AANATL2 by ESI-MS    45
Figure 3.4 Purification of AANATL2 by gel filtration column    46
Figure 3.5 Crystals of AANATL2 screening crystal kit    47
Figure 3.6 Substrate screening    48
Figure 3.7 DTNB functional assay for AANATL2    49
Figure 3.8 ITC study for binding affinity and thermodynamics of AANATL2 with Ac-CoA and substrate PEA    50
Figure 3.9 The merged ITC profiles of ligands binding    51
Figure 3.10 The sequence alignment of AANATL2 and Dat    52
Figure 3.11 Modelling Structure of AANATL2    53
Figure 3.12 Modeling structure align with Dat ternary form    54
Figure 3.13 Docking results of Ac-CoA and PEA into modeling structure of AANATL2    55
Figure 3.14 Investigation into the Ac-CoA and PEA binding site of AANATL2 by Ligplot+    56
Figure 3.15 Seven mutated candidates in AANATL2 modelling structure    57
Figure 3.16 The PCR results of mutants    58
Figure 3.17 Purification of AANATL2 mutants by Ni2+ -affinity column    59
Figure 3.18 Purification of AANATL2 mutants by Ni2+ -affinity column    60
Figure 3.19 Purification of AANATL2 mutants by Ni2+ -affinity column    61
Figure 3.20 Purification of AANATL2 mutants by Ni2+ -affinity column    62
Figure 3.21 The secondary structure of AANATL2 and mutants    63
Figure 3.22 DTNB-based functional assay of L2-WT and mutants    64
Figure 3.23 ITC study for Ac-CoA/PEA binding affinity and thermodynamics of AANATL2 (as L2-WT) and mutant    65
Figure 3.24 ITC study for Ac-CoA/PEA binding affinity and thermodynamics of mutant    66
Figure 3.25 ITC study for PEA/Ac-CoA binding affinity and thermodynamics of AANATL2 (as L2-WT) and mutant    67
Figure 3.26 ITC study for PEA/Ac-CoA binding affinity and thermodynamics of AANATL2 (as L2-WT) and mutant    68
Supplementary Table 3.1 Kinetic parameters of AANATLs and Dat    69
Supplementary Figure 3.5 The sequencing alignment of mutants    73
Supplementary Figure 3.6 The sequencing alignment of mutants    74
Reference    75
                                

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