- 學位論文
果蠅多巴胺乙醯基轉移酶功能與結構之研究
Structural and functional studies of dopamine N-acetyltransferase from Drosophila melanogaster
摘要
褪黑激素(melatonin)是一個主要受光誘導隨晝夜光週期變化的生物荷爾蒙調解因子，並廣泛分布於細菌，原生動物，海藻，植物，真菌，無脊椎動物和脊椎動物。隨著褪黑激素的分子結構已經被理解，這個“分子韻律”激素引起廣泛研究興趣。褪黑激素的生理作用已被研究與睡眠，情緒，免疫反應，心血管功能和老化有關。褪黑激素的生物合成研究可以幫助深入了解晝夜周期的機制。在哺乳動物體內的褪黑激素的合成是受到苯烷基胺乙醯基轉移酶（AANAT; EC 2.3.1.87）依晝夜週期節律所調控，致使此酵素成為發展與情緒和睡眠障礙的治療作用的藥物標的。果蠅的多巴胺乙醯基轉移酶(dopamine N-acetyltransferase; Dat)是一個苯烷基胺乙醯基轉移酶（arylalkylamine N-acetyltransferase AANAT; EC 2.3.1.87），這是涉及褪黑激素的生產、昆蟲表殼硬化與神經傳導物質去活化之酵素並已發現存在於果蠅的頭部、眼睛、視葉和大腦。此外，多巴胺乙醯基轉移酶屬於GCN5相關乙醯基轉移酶（GNAT）家族的成員。多巴胺乙醯基轉移酶催化乙醯輔酶A (acetyl coenzyme A/輔因子)的乙醯基轉移到各種苯烷基胺(arylalkylamine/基質)。 為了深入探究褪黑激素合成之分子機制，我們著手於多巴胺乙醯基轉移酶的結構和功能的研究。我們已經成功解出果蠅多巴胺乙醯基轉移酶之不同高解析度結構:包括單元、 双元(乙醯輔酶A複合)、 三元 (乙醯苯烷基胺/輔酶A複合)之晶體結構。由利用等溫滴定微量熱法（ITC）來探討酵素、基質與輔因子之間的結合作用力的研究中結果暗示輔助因子能夠優先與酵素結合。藉由分析複合晶體結構與基質分子對接模型(docking model)結果顯示多巴胺乙醯基轉移酶在活性部位具有獨特的催化三元體(catalytic triad)。結合定點突變與酵素動力學的研究結果證實Glu47、Ser182和Ser186扮演重要催化角色。我們的研究結果證實，果蠅多巴胺乙醯基轉移酶擁有一個特殊的催化反應活性中心並利用催化三元體(catalytic triad)胺基酸經由促進苯烷基胺的氨基直接對乙醯輔酶A中的乙醯羰基作親核攻擊並且促使輔酶的烴硫基離子氫化來進行催化反應。 目前關於多巴胺乙醯基轉移酶以分子為基礎的基質辨識和酵素分子、基質與輔助因子之間反應動力學機制尚不清楚。基質/輔因子與其類似物抑制劑(色醇和棕櫚醯輔酶A)之酵素動力學分析結果顯示多巴胺乙醯基轉移酶是以輔助因子優先與酵素結合的順序型反應機制進行催化反應。再者，我們提出的第一個苯烷基胺乙醯基轉移酶家族之三元複合物的晶體結構。詳細分析巴胺乙醯基轉移酶之三元複合物的晶體結構，顯示在活性位置附近具有一個疏水性基質接合空腔。此疏水性空腔的形狀與大小決定基質的選擇性與專一性。我們已經發現多巴胺乙醯基轉移酶在基質接合空腔具有兩個重要芳香族殘基對於基質接合與苯烷基胺、吲哚烷基胺、芳香胺基質之間的選擇扮演重要角色。多巴胺乙醯基轉移酶對於苯烷基胺具有較高的活性。苯烷基胺之芳香環和烷基鏈的長度影響多巴胺乙醯基轉移酶催化基質的活性。 結合三元複合物結構分析與定點突變的研究，Phe43 此殘基被證實在酵素與基質接合扮演重要角色; Tyr64 影響酵素的基質選擇性。動力學實驗證實，Y64W突變體足以增加酵素對於吲哚烷基胺的活性，這表明該殘基調控多巴胺乙醯基轉移酶對於苯烷基胺與吲哚烷基胺之間的基質偏好選擇性。這些結果證實，這些殘基提供芳香環作用力，此作用力在酵素和基質之間的相互作用中扮演關鍵角色。 本研究提供了一個結構的基礎來幫助瞭解苯烷基胺乙醯基轉移酶和一般GCN5相關乙醯基轉移酶（GNAT）家族的結構和生物特性。
並列摘要
Melatonin is a major hormonal mediator of light-induced photoperiodic changes in circadian biological events and is found in bacteria, protozoa, macroalgae, plants, fungi, invertebrates, and vertebrates. Despite of the structural characterization of melatonin, there has been increasing interest in this “molecular pacemaker” hormone. The physiological roles of melatonin have been widely reported on sleep, mood, immune response, cardiovascular fitness and aging. Research on melatonin biosynthesis could help improve our knowledge of circadian rhythm. The daily cycle of melatonin biosynthesis in mammals is regulated by AANAT (arylalkylamine N-acetyltransferase; EC 2.3.1.87), making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. Drosophila melanogaster Dat (dopamine N-acetyltransferase) is an arylalkylamine N-acetyltransferase, which is involved in melatonin formation, sclerotization, and neurotransmitter inactivation and has been found in the head, the eyes, the optic lobe and the brain of Drosophila melanogaster. Moreover, Dat belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. Dat catalyzes the transfer of the acetyl group in acetyl coenzyme A (AcCoA, cofactor) to various arylalkylamines (substrate). In order to unravel the detail molecular mechanism for Dat activity, we worked on the structural and functional studies of Dat. We have determined high-resolution crystal structure of D. melanogaster Dat in apo form, binary complex form (AcCoA-bound), and ternary complex form (acetylarylalkylamine/CoA-bound). A binding study using isothermal titration calorimetry suggested that the cofactor bound to Dat first before substrate. Examination of the binary complex structure and a substrate-docked model indicated that Dat contains a novel AANAT catalytic triad. Site-directed mutagenesis, kinetic studies and pH-rate profiles confirmed that Glu47, Ser182 and Ser186 were critical for catalysis. Collectively, the results of the present study suggest that Dat possesses a specialized active site structure dedicated to a catalytic mechanism where nucleophilic attack and leaving group protonation occur in a coordinated manner dependent on catalytic triad. The molecular basis of substrate recognition and the kinetic mechanism by which Dat interacts with substrate and cofactor are unclear. Here, two-substrate kinetic analysis and dead end analog inhibition studies with the tryptophol and palmitoyl CoA indicated that Dat utilizes an ordered sequential mechanism requiring binding of acetyl-CoA first. Furthermore, we presented the first crystal structure of ternary complex in this AANAT family. Detailed analyses of ternary complexes of Dat revealed a hydrophobic substrate-binding pocket near the acetylation active site. The shape and size of the pocket dictate substrate selectivity and specificity. We have mapped two key aromatic residues in the protein-substrate interface essential for substrate binding and selection between phenylalkylamines (PAAs), indoalkylamines (IAAs), and arylamines substrates. The Dat has higher activity with the PAAs than with the IAAs. It appears that the aromatic ring and alkyl chain length on arylalkylamine molecule greatly define the Dat extended substrate specificity profile. By analyzing ternary complex structure as well as site-directed mutagenesis, we demonstrated that Phe43 significantly influence the substrate binding and the activity of Dat, while Tyr64 was an important determinant of substrate preference. Kinetic studies confirmed that the Y64W mutation is sufficient to increase the activity of the enzyme toward IAAs being the preferred substrate for the Y64W mutant, which indicated that this residue modulates the substrate preference of Dat between PAAs and IAAs. These results confirmed that these residues are critical for aromatic interaction between Dat and substrate. This work provides a structural foundation for the detailed understanding of the structural and biological properties of arylalkylamine N-acetyltransferases and of GCN5-related N-acetyltransferase superfamily proteins in general.
參考文獻
延伸閱讀
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