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標題: | 斑馬魚中亞精胺/精胺 N1-乙醯基轉移酵素之結構與功能分析:受質辨認機制探討 Structure and functional analysis of zebrafish spermidine/spermine N1-acetyltransferase: insight into the substrate recognition |
作者: | Yi-Ruei Chang 張宜叡 |
指導教授: | 徐駿森(Chun-Hua Hsu) |
關鍵字: | 精胺,亞精胺,腐胺,亞精胺/精胺 N1-乙醯基轉移酵素,蛋白質結晶學,恆溫滴定微量熱儀, Spermine,spermidine,putrescine,spermidine/spermine N1-acetyltransferase,protein crystallography,crystal structure, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 多胺 (Polyamines, PA) 是一類擁有兩個或多個一級胺基的有機化合物,如腐胺 (Putrescine)、亞精胺 (Spermidine) 和精胺 (Spermine),廣泛地存在於各物種並對於細胞的生存扮演非常重要的角色,因此 PA 在細胞中必須維持動態平衡,而亞精胺/精胺 N1-乙醯基轉移酵素 (Spermidine/spermine N1-acetyltransferase, Ssat) 則為合成和代謝途徑調控的關鍵酵素。在斑馬魚中有三種 zSsat1s (1a、1b、1c) 和兩種 zSsat2s (2a、2b),並有著不同的受質選擇性。因此我們希望藉由比較酵素結構的差異,進而闡述其不同受質的分子辨識模式。經表現純化此五種 zSsats 的重組蛋白,藉由酵素活性實驗與恆溫滴定微量熱儀的量測,比較出酵素對於不同受質之選擇性,利用蛋白質晶體學獲得 zSsat1a 的晶體且收集到 2.61A 之解析度,經由結構上的觀察與比較,發現了數個可能影響受質選擇性的熱點 (hotspot)。此外,zSsat 在細胞內以同源雙聚體存在,而這些不同的異構體亦會形成異源雙聚體,並發揮有別於同源雙聚體的活性。為證實此推論,我們將兩種 zSsat2 (2a、2b) 混合後測酵素活性,意外發現對原本非受質的 putrescine 產生催化。為模擬異源雙聚體所形成之受質結合區域,於是將兩種異構基因的 N 端和 C 端做嵌合互換後,發現其中一種 zSsat2b2a chimera 對 putrescine 也有活性,此酵素也已被獲得晶體並收集到 2.65A 之解析度,配合酵素活性與 ITC 實驗,發現 zSsat2b2a chimera 在受質辨識的熱點 (hotspot) 與 CoA 的結合位置皆有所不同,因此我們認為酵素可以藉由形成異源雙聚體而產生新的受質辨識區域。 Polyamines (PAs) are organic compound with two of more primary amino groups (such as putrescine, spermidine and spermine), which play vital roles of cells and widely found in the all kinds of species. Spermidine/spermine N1-acetyltransferase (Ssat) is a key enzyme, which maintains PA homeostasis. However, there exist three zSsat1s (1a, 1b, 1c) and two zSsat2s (2a, 2b) in zebrafish, which possess different specificities to the PAs. In order to understand the substrate recognition, biochemical and structural elucidations of these zSsats were performed. We have expressed five zSsats from the cloned cDNA in E. coli and have determined structure of zSsat1a at resolution of 2.61 Ǻ. Comprehensive ITC and enzymatic assays of zSsats were further investigated. By sequence and structure comparison among the zSsats, a number of hotspots involved in the substrate recognition were found. In addition, we demonstrate by enzymatic assays and ITC that zSsat2a exists as a heterodimer with zSsat2b. Surprisingly, heterodimer exerts novel substrate specificity for putrescine, which is different from homodimers’ preferences. Two chimeras with N- and C-terminus exchange of zSsat2s were then created and named as zSsat2a2b and zSsat2b2a. Functionally, the zSsat2b2a possess the unexpected activity for putrescine. Crystal structure of zSsat2b2a was solved to 2.65 A. Hotspots in zSsat2b2a were different, and changed the specificity of the substrate, providing a new mode of substrate binding. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60859 |
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顯示於系所單位: | 農業化學系 |
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