臺灣博碩士論文加值系統

10-甲醛四氫葉酸去氫酶是在葉酸單碳循環中的一個酵素，主要催化的反應是將10-甲醛四氫葉酸轉變成四氫葉酸及二氧化碳，在靈長類的甲醇中毒中是唯一可以把甲酸鹽氧化的路徑。目前研究過的哺乳類10-甲醛四氫葉酸去氫酶是以同型四聚體形式存在。而每一個單元體約99 kDa。每一個單元體是由一個小的N端區域及一個大的C端區域所構成的。在N端區域具有10-甲醛四氫葉酸水解酶的活性。C端區域具有短鏈醛類去氫酶的活性。當N端及C端都位在正確的相對位置時，才具有10-甲醛四氫葉酸去氫酶的酵素活性。到目前為止，有數種10-甲醛四氫葉酸去氫酶可能的生理功能被提出來。包括：調節還原態的葉酸、儲存細胞內葉酸、調節（de novo）嘌呤的生合成與細胞內的甲基化反應。
我們從斑馬魚的cDNA基因庫中將10-甲醛四氫葉酸去氫酶選殖出，並在E.coli 及Pichia的系統中表現及純化。所得之蛋白質皆有大於90%的純度，但都只能測到N端的水解酶活性。此10-甲醛四氫葉酸去氫酶的三級及四級結構和兔子與人類的同功酶很相似，具有30 kDa的N端與形成四聚體的C端。分別選殖N端及C端的10-甲醛四氫葉酸去氫酶則各具有水解酶及短鏈醛類去氫酶的酵素活性。將10-甲醛四氫葉酸去氫酶送入HEK 293T 選殖穩定表現細胞株進行蛋白質表現，則發現表現出來斑馬魚的10-甲醛四氫葉酸去氫酶具有10-甲醛四氫葉酸去氫酶的酵素活性。在針對10-甲醛四氫葉酸去氫酶的組織專一性分佈的研究中發現，斑馬魚10-甲醛四氫葉酸去氫酶主要表現在肝臟，與哺乳類動物一致。綜合所有結果我們認為斑馬魚應可作為葉酸代謝循環研究的動物模型。

10-Formyltetrahydrofolate dehydrogenase(FDH), a folate-requiring enzyme in one-carbon metabolism, catalyzes the NADP+-dependent oxidation of 10-formyltetrahydrofolate (10-CHO- H4PteGlu) to form CO2 and tetrahydrofolate. This is the the only pathway responsible for formate oxidation in methanol intoxication in primates. Mammalian FDH is a homotetramer composed of 99 kDa subunits. Each monomer consists of a small N-terminal domain and a big C-terminal domain. The N-terminal domain catalyzes only the NADP+-independent 10-CHO-H4PteGlu hydrolase reaction and the C-terminal domain catalyzes only the NADP+-dependent aldehyde dehydrogenase reaction, whereas the 10-CHO-H4PteGlu dehydrogenase activity requires the presence of both N- and C-terminal domains situated in the appropriate orientation.
The aim of this study is to functionally and structurally characterize FDH from zebrafish, a prominent animal model for basic research and drug screening. We have cloned and purified the full-length FDH overexpressed in Escherichia coli Rosetta (DE3) cells and Pichia Pastoris. Size exclusion chromatography and limited digestion with subtilisin suggest strong similarity in their tertiary and quaternary structures between the recombinant zFDH and mammalian orthologs. However, the purified enzyme is only partially active with measurable 10-CHO-H4PteGlu hydrolase activity. Subcloning and expression of both zFDH N-terminal and C-terminal domains separately in E.coli result in fully functional 10-CHO-H4PteGlu hydrolase and aldehyde dehydrogenase activities, respectively. The full-length zFDH was also cloned to pTracer and expressed in human kidney 293T cells, and 10-formyltetrahydrofolate dehydrogenase activity corresponding to the transfected zFDH was detected in the cell extract. The estimated apparent kcat of 10-CHO-H4PteGlu dehydrogenase activity is 461min-1 ,which is similar to that of mammalian orthologs. Tissue-specific distribution was observed with the highest expression present in liver. The similarity revealed in our results for zFDH compared to mammalian orthologs suggests that zebrafish could be an appropriate model for folate-mediated one carbon metabolism.

一、 圖目錄……………………………2~3
二、 表目錄……………………………………4
三、 緒論………………………5~11
四、 研究材料及方法…………………12~25
五、 結果………………………………26~31
六、 討論…………………………32~35
七、 參考文獻……………………36~40
八、 圖………………………………41~63
九、 表格………………………………64~67
十、 附件……………………………68~79
十一、 自述………………………………80

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