在哺乳动物的表观遗传调控中,TET蛋白参与了DNA的主动去甲基化过程,该蛋白催化产生的5-羧基胞嘧啶可能通过两种途径被转变为胞嘧啶,其中一种途径就是通过潜在的DNA脱羧酶直接催化5-羧基胞嘧啶发生脱羧反应。由于胞嘧啶和尿嘧啶的化学结构和性质比较相似,因此,潜在的DNA脱羧酶可能与真菌中尿嘧啶脱羧酶IDCase在结构和催化机制上存在一定的相似性。
2013年8月6日国际知名杂志《细胞研究》 (Cell Research)在线发表了生物化学与细胞生物学研究所丁建平组关于尿嘧啶脱羧酶的催化机制的最新研究成果,揭示了IDCase的催化机制,为寻找哺乳动物中潜在的DNA脱羧酶提供了新思路。
DNA胞嘧啶的甲基化是一种重要的表观遗传修饰,在很多生物学过程中都发挥重要作用。DNA胞嘧啶的甲基化修饰由DNA甲基转移酶催化发生,然而DNA的主动去甲基化是如何发生的却长期未有定论。近期研究表明,TET蛋白通过逐步氧化5-甲基胞嘧啶生成5-羧基胞嘧啶,且可能存在DNA脱羧酶直接催化5-羧基胞嘧啶脱羧生成无修饰的胞嘧啶。胞嘧啶和尿嘧啶的结构非常相似,此外哺乳动物中TET蛋白催化5-甲基胞嘧啶发生三步氧化反应最终生成5-羧基胞嘧啶的过程与真菌中T7H催化胸腺嘧啶转变为5-羧基尿嘧啶的过程极为相似。鉴于此,哺乳动物中很有可能存在DNA脱羧酶,而这种潜在的DNA脱羧酶与真菌中催化5-羧基尿嘧啶转变为尿嘧啶的IDCase在序列、整体结构、底物结合和催化机制等方面可能存在较多相似性。此外,IDCase的底物识别和催化反应机制也未有报道。
丁建平研究组的博士生徐曙彤和李文婧等人解析了Cordyceps militaris来源的尿嘧啶脱羧酶IDCase (CmIDCase)的野生型和突变体原酶(apo)形式以及与底物5-羧基尿嘧啶、底物类似物5-硝基尿嘧啶和产物尿嘧啶的复合物的晶体结构,以及Metarhizium anisopliae来源的IDCase的原酶形式的晶体结构。结构分析表明,IDCase呈现典型的氨基水解酶超家族所含有的(β/α)8桶状折叠结构,在原酶形式和结合配体形式的CmIDCase结构中,活性位点处都结合了一个Zn2+。进一步的突变体和酶活实验验证了参与金属离子结合和底物结合的关键氨基酸在催化反应中的功能。基于结构分析和体外生化实验结果,提出了一种新的脱羧反应催化机制。他们的研究结果不仅揭示了尿嘧啶脱羧酶IDCases的底物识别和催化机制,并为寻找哺乳动物中潜在的DNA脱羧酶提供了新思路和重要信息。
该项研究工作得到了生化与细胞所徐国良研究员的帮助和国家科技部、国家自然科学基金委、上海市科委的经费支持。(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.