在哺乳動(dòng)物的表觀遺傳調(diào)控中,，TET蛋白參與了DNA的主動(dòng)去甲基化過程,，該蛋白催化產(chǎn)生的5-羧基胞嘧啶可能通過兩種途徑被轉(zhuǎn)變?yōu)榘奏ぃ渲幸环N途徑就是通過潛在的DNA脫羧酶直接催化5-羧基胞嘧啶發(fā)生脫羧反應(yīng)。由于胞嘧啶和尿嘧啶的化學(xué)結(jié)構(gòu)和性質(zhì)比較相似,，因此,，潛在的DNA脫羧酶可能與真菌中尿嘧啶脫羧酶IDCase在結(jié)構(gòu)和催化機(jī)制上存在一定的相似性。
2013年8月6日國際知名雜志《細(xì)胞研究》 (Cell Research)在線發(fā)表了生物化學(xué)與細(xì)胞生物學(xué)研究所丁建平組關(guān)于尿嘧啶脫羧酶的催化機(jī)制的最新研究成果,，揭示了IDCase的催化機(jī)制,，為尋找哺乳動(dòng)物中潛在的DNA脫羧酶提供了新思路。
DNA胞嘧啶的甲基化是一種重要的表觀遺傳修飾,，在很多生物學(xué)過程中都發(fā)揮重要作用,。DNA胞嘧啶的甲基化修飾由DNA甲基轉(zhuǎn)移酶催化發(fā)生，然而DNA的主動(dòng)去甲基化是如何發(fā)生的卻長(zhǎng)期未有定論,。近期研究表明,，TET蛋白通過逐步氧化5-甲基胞嘧啶生成5-羧基胞嘧啶，且可能存在DNA脫羧酶直接催化5-羧基胞嘧啶脫羧生成無修飾的胞嘧啶,。胞嘧啶和尿嘧啶的結(jié)構(gòu)非常相似,，此外哺乳動(dòng)物中TET蛋白催化5-甲基胞嘧啶發(fā)生三步氧化反應(yīng)最終生成5-羧基胞嘧啶的過程與真菌中T7H催化胸腺嘧啶轉(zhuǎn)變?yōu)?-羧基尿嘧啶的過程極為相似。鑒于此,，哺乳動(dòng)物中很有可能存在DNA脫羧酶,，而這種潛在的DNA脫羧酶與真菌中催化5-羧基尿嘧啶轉(zhuǎn)變?yōu)槟蜞奏さ腎DCase在序列、整體結(jié)構(gòu),、底物結(jié)合和催化機(jī)制等方面可能存在較多相似性,。此外，IDCase的底物識(shí)別和催化反應(yīng)機(jī)制也未有報(bào)道,。
丁建平研究組的博士生徐曙彤和李文婧等人解析了Cordyceps militaris來源的尿嘧啶脫羧酶IDCase (CmIDCase)的野生型和突變體原酶（apo）形式以及與底物5-羧基尿嘧啶,、底物類似物5-硝基尿嘧啶和產(chǎn)物尿嘧啶的復(fù)合物的晶體結(jié)構(gòu)，以及Metarhizium anisopliae來源的IDCase的原酶形式的晶體結(jié)構(gòu),。結(jié)構(gòu)分析表明,，IDCase呈現(xiàn)典型的氨基水解酶超家族所含有的(β/α)8桶狀折疊結(jié)構(gòu)，在原酶形式和結(jié)合配體形式的CmIDCase結(jié)構(gòu)中,，活性位點(diǎn)處都結(jié)合了一個(gè)Zn2+,。進(jìn)一步的突變體和酶活實(shí)驗(yàn)驗(yàn)證了參與金屬離子結(jié)合和底物結(jié)合的關(guān)鍵氨基酸在催化反應(yīng)中的功能?；诮Y(jié)構(gòu)分析和體外生化實(shí)驗(yàn)結(jié)果,，提出了一種新的脫羧反應(yīng)催化機(jī)制。他們的研究結(jié)果不僅揭示了尿嘧啶脫羧酶IDCases的底物識(shí)別和催化機(jī)制,，并為尋找哺乳動(dòng)物中潛在的DNA脫羧酶提供了新思路和重要信息,。
該項(xiàng)研究工作得到了生化與細(xì)胞所徐國良研究員的幫助和國家科技部、國家自然科學(xué)基金委,、上海市科委的經(jīng)費(fèi)支持,。（生物谷Bioon.com）
doi:10.1038/cr.2013.107
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Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase.
Shutong Xu, Wenjing Li, Junjun Zhu, Rong Wang, Zheng Li, Guo-Liang Xu and Jianping Ding
DNA methylation and demethylation regulate many crucial biological processes in mammals and are linked to many diseases. Active DNA demethylation is believed to be catalyzed by TET proteins and a putative DNA decarboxylase that may share some similarities in sequence, structure and catalytic mechanism with isoorotate decarboxylase (IDCase) that catalyzes decarboxylation of 5caU to U in fungi. We report here the structures of wild-type and mutant IDCases from Cordyceps militaris and Metarhizium anisopliae in apo form or in complexes with 5caU, U, and an inhibitor 5-nitro-uracil. IDCases adopt a typical (β/α)8 barrel fold of the amidohydrolase superfamily and function as dimers. A Zn2+ is bound at the active site and coordinated by four strictly conserved residues, one Asp and three His. The substrate is recognized by several strictly conserved residues. The functional roles of the key residues at the active site are validated by mutagenesis and biochemical studies. Based on the structural and biochemical data, we present for the first time a novel catalytic mechanism of decarboxylation for IDCases, which might also apply to other members of the amidohydrolase superfamily. In addition, our biochemical data show that IDCases can catalyze decarboxylation of 5caC to C albeit with weak activity, which is the first in vitro evidence for direct decarboxylation of 5caC to C by an enzyme. These findings are valuable in the identification of potential DNA decarboxylase in mammals.
