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

許育碩(Yu-Shuo Hsu)

指導教授 ： 呂平江

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2021年)

摘要

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。

關鍵字

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

並列摘要

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.

並列關鍵字

N-acetyltransferase ； acetyl-coenzymeA ； X-ray crystallography ； arylalkylamine N-acetyltransferase ； agmatine ； ITC

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