Structural studies of the sarcosine dimethylglycine methyltransferase from Methanohalophilus portucalensis FDF1T, an enzyme involved in betaine biosynthesis

甲基轉移酶廣泛的參與各種重要的生物功能，如biosynthesis、metabolism、detoxification、signal transduction、protein sorting 與 repair，以及 nucleic acid processing等等…。也因此各種生物體皆擁有各式各樣具備不同受質專一性與調控特性的甲基轉移酶，其中又以使用S-Adenosyl-L-Methionine (SAM) 作為甲基提供者的SAM- dependent甲基轉移酶最為普遍。根據SAM-binding domain之結構，可將SAM-dependent甲基轉移酶區分為五類 ( class I~V )，其中大多數都屬於class I，這些甲基轉移酶在催化反應時遵循SN2反應機制，酵素活性的高低則取決於甲基受體的親核性強弱與酵素活性中心是否能有效穩定反應過渡狀態以降低活化能。以class I SAM- dependent甲基轉移酶為例，結構分析指出有些N-或C-甲基轉移酶的活性中心具有可增強甲基受體親核性之高保留性motif或胺基酸，例如DDPY motif、active site cysteine、H-Y general base等。但尚未有明確報導指出class I SAM-dependent甲基轉移酶之活性中心具有可穩定反應過渡狀態的結構存在。本篇研究解析出屬於class I的sarcosine dimethyl- glycine N-methyltransferase (SDMT) 之apo form、SAM-bound，以及Sar/SAH-bound等不同狀態之結構，並且發現SDMT可藉由SAM和受質結合所產生的構形變化，逐步在甲基轉移路徑上形成一個由三個氧原子組成之三角平面結構 (稱為三氧模組)。當胺基酸突變去除或改變三氧模組上任一氧原子的位置時，都會造成催化活性大幅下降。以Density Functional Theory計算SDMT催化甲基轉移反應的自由能變化可知當此三氧模組存在時確實會降低活化能，推論三氧模組應可穩定甲基轉移反應時的過渡狀態。另外也嘗試透過結構比對分析此三氧模組於SAM-dependent甲基轉移酶活性中心出現的普遍性。結果顯示此三氧模組僅存在於甲基受體為經由去質子化呈現電中性之氮原子的N-甲基轉移酶，或是以雙鍵上?電子做為親核基之C-甲基轉移酶。而在甲基受體為去質子化後帶負電之氧原子的O-甲基轉移酶則無此結構。此結果暗示了甲基轉移酶是否具有三氧模組極可能與甲基受體親核性強弱有關。這是首次揭示class I SAM-dependent甲基轉移酶可藉由逐步的構形變化在活性中心組合出一個具催化活性的三氧模組結構，為甲基轉移酶之異位調控機制提供新的想法。In biological systems, methylation reactions are involved in a wide variety of functions, including biosynthesis, metabolism, detoxification, signal transduction, protein sorting and repair, and nucleic acid processing. A large number of methyltransferases (MTs) have been identified to act on distinct substrates. Most MTs perform substrate methylation by using S-adenosyl-methionine (SAM or AdoMet) as the methyl group donor. and the reactions catalyzed by these 'SAM-dependent MTs' usually proceed via an nucleophilic substitution (SN2) mechanism, with the atom targeted for methylation acting as the nucleophile. By employing 'proximity and desolvation effects', the 'acid-base' or a metal-dependent mechanism, MTs facilitate reaction by enhancing the nucleophilicity of the atom targeted for methylation. Furthermore, the involvement of a conserved DPPY motif and the active site cysteine has also been identified in nucleotide base or protein MTs. In this study, we have revealed the previously unrecognized catalytic role of a tri-oxygen module in the active site of the sarcosine dimethylglycine N-methyl-transferase (SDMT) from Methanohalophilus portucaensis. Specifically, crystal structures of SDMT, alone and in complexes with substrate, inhibitor, or the cofactor SAM have been determined at high resolution. Structural comparison revealed large conformational change induced by the binding of substrate and cofactor, which results in the formation of a substrate-binding and a cofactor-binding pocket connected by a narrow channel. This pocket-connecting channel is shaped in the middle by three triangularly arranged oxygen atoms, constituted by the phenolic oxygen of Y16 and the two main-chain carbonyl groups of I34 and D142. By lining halfway between SAH and substrate, it appears that this tri-oxygen module are positioned to stabilize the transition state of SN2-like reaction to promote transfer of methyl group. Consistent with this hypothesis, the kcat value of Y16F, I34A and D142A mutants dropped to about 10%. Besides, density functional theory (DFT) calculations show the tri-oxygen module may indeed stabilize the SN2 reaction transition state for N-methylation. Importantly, we noted that this tri-oxygen module is present in those N- and C-methyltransferases whose methyl group acceptors are neutral nitrogen atoms and ?-electron-containing carbon atoms, respectively. In contrast, O-methyltransferases do not possess such a module, suggesting its presence may correlate with the nucleophilicity of the methyl group accepting atom. This is the first study to reveal that, through conformational changes, Class I SAM-dependent MTases can assemble catalytic tri-oxygen modules in the active sites. Based on the finding of such a catalysis mechanism, this study provided new implications on the potential allosteric regulation of MTases.目錄 摘要………………………………………………………………………I Abstract………………………………………………………………….III 目錄…………………………………………………………………...…V 表目錄………………………………………………………………...VIII 圖目錄…………………………………………………………………..IX 壹、 前言………………………………………………………………..1 貳、 材料與方法………………………………………………………..5 一、 菌株與質體…………………………………………………......5 二、 菌種之保存……………………………………………………..5 三、 蛋白質大量表現………………………………………………..5 四、 [Se-Met]MpSDMT蛋白質樣品製備…………………………..6 五、 MpSDMT與[Se-Met]MpSDMT以及突變株蛋白質的萃取與純化………………………………………………………………..6 ( 1 ) 破菌……………………………………………………….6 ( 2 ) 親和性管柱（Nickel column）初步純化………………..7 ( 3 ) 濃縮……………………………………………………….7 ( 4 ) 分子篩管柱純化………………………………………….8 六、 蛋白質養晶前處理與結晶步驟………………………………..8 ( 1 ) 蛋白質濃縮、定量………………………………………..8 ( 2 ) 養晶……………………………………………………….8 ( 3 ) 微調結晶條件……………………………….....................9 七、 MpSDMT與[Se-Met]MpSDMT蛋白X-ray繞射實驗..............9 ( 1 ) X-ray繞射前準備................................................................9 ( 2 ) 晶體繞射數據收集及分析...............................................10 八、 MpSDMT與受質及抑制劑（SAH）之共結晶實驗和繞射數據分析............................................................................................11 ( 1 ) MpSDMT與受質及SAH之共結晶實驗.........................11 ( 2 ) 晶體繞射數據收集及分析...............................................11 九、 MpSDMT與輔酶(SAM)之共結晶實驗和繞射數據分析........11 ( 1 ) MpSDMT與SAM之共結晶實驗....................................11 ( 2 ) 晶體繞射數據收集及分析...............................................12 十、 MpSDMT催化反應之中間產物累積實驗...............................12 十一、 甲基轉移酶活性分析..........................................................12 十二、 Density Functional Theory ( DFT ) 理論計算...................13 參、 結果與討論....................................................................................15 一、 MpSDMT親和性管柱純化.......................................................15 二、 分子篩管柱純化........................................................................15 三、 結晶............................................................................................15 四、 X-ray繞射數據的分析與unit cell大小判定............................16 五、 蛋白晶體結構建立....................................................................16 六、 受質與輔酶 (cofactors) 的結合造成SDMT顯著的結構變化 (conformational change)............................................................17 七、 SAM結合所誘發的結構變化可能與促進受質結合有關.......19 八、 受質結合誘發構形變化形成封閉的疏水性活性中心............20 九、 活性中心水分子之作用............................................................21 十、 結構上達成受質與產物專一性的可能機制............................21 十一、 三個氧原子組成之三角平面結構的催化角色.................22 十二、 Density Functional Theory (DFT) 理論計算結果.............24 十三、 SAM-dependent N-甲基轉移酶是否具有三氧模組結構與甲基受體的親核性強弱有關....................................................24 肆、 結論與展望....................................................................................25 伍、 圖與表............................................................................................27 陸、 參考文獻........................................................................................5