Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92213
標題: 高鹽甲烷太古生物之相容質甜菜鹼自體生合成酵素-肌胺酸、二甲基甘胺酸甲基轉移酶的結構與功能研究
Structural studies of the sarcosine dimethylglycine methyltransferase from Methanohalophilus portucalensis FDF1T, an enzyme involved in betaine biosynthesis
作者: 林德昇
Te-Sheng Lin
關鍵字: 甲基轉移酶;三氧模組;甜菜鹼;SAM-MT;tri-oxygen module;betaine
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摘要: 
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.
URI: http://hdl.handle.net/11455/92213
Rights: 同意授權瀏覽/列印電子全文服務,2018-01-27起公開。
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