A bifunctional dCTP deaminase-dUTP nucleotidohydrolase from the hyperthermophilic archaeon Methanocaldococcus jannaschii
A bifunctional dCTP deaminase-dUTP nucleotidohydrolase from the hyperthermophilic archaeon Methanocaldococcus jannaschii
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DOI：
10.1074/jbc.m213010200
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发表时间：
2003-06-06
影响因子：
4.8
通讯作者：
Nyman, PO
中科院分区：
文献类型：
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作者：
Björnberg, O;Neuhard, J;Nyman, PO
通过dCTP脱氨酶和dutp酶的连续作用，dCTP转化为dUMP，这是胸腺嘧啶核苷酸的前体。此外，dUTPase在防止尿嘧啶掺入DNA方面具有重要作用。大肠杆菌中过量产生了来自超嗜热古细菌jannaschii甲烷醛球菌的dCTP脱氨酶（MJ0430）和dutp酶（MJ1102）。出乎意料的是，我们发现MJ0430蛋白能够进行这两种反应，即胞嘧啶环的水解脱氨和α -磷酸和β -磷酸之间的磷酸酐键的水解裂解。当以[H-3] dCTP为底物进行薄层色谱反应后，确定了dUMP而不是dUTP为反应产物。当未标记的dUTP作为抑制剂存在时，没有标签从[H-3] dCTP转移到dUTP池中。这一发现有力地表明，反应的两个连续步骤在酶内是紧密耦合的。迄今为止未知的MJ0430蛋白的双重功能似乎对细胞有益，因为有毒的中间dUTP从未释放。MJ0430蛋白也能催化dUTP水解为dUMP，但对底物的亲和力较低（K-m>100 muM）。根据有限的蛋白水解，c端残基构成一个柔性区域。另一种被研究的蛋白MJ1102是一种特异性dUTP酶，其dUTP的K-m （0.4 muM）的大小与先前发现的dUTP酶相当。其生理功能可能是降解核苷酸代谢中其他反应产生的dUTP。
By the sequential action of dCTP deaminase and dUTPase, dCTP is converted to dUMP, the precursor of thymidine nucleotides. In addition, dUTPase has an essential role as a safeguard against uracil incorporation in DNA. The putative dCTP deaminase (MJ0430) and dUTPase (MJ1102) from the hyperthermophilic archaeon Methanocaldococcus jannaschii were overproduced in Escherichia coli. Unexpectedly, we found the MJ0430 protein capable of both reactions, i.e. hydrolytic deamination of the cytosine ring and hydrolytic cleavage of the phosphoanhydride bond between the alpha- and beta-phosphates. When the reaction was followed by thin layer chromatography using [H-3] dCTP as substrate, dUMP and not dUTP was identified as a reaction product. In the presence of unlabeled dUTP, which acted as an inhibitor, no label was transferred from [H-3] dCTP to the pool of dUTP. This finding strongly suggests that the two consecutive steps of the reaction are tightly coupled within the enzyme. The hitherto unknown bifunctionality of the MJ0430 protein appears beneficial for the cells because the toxic intermediate dUTP is never released. The MJ0430 protein also catalyzed the hydrolysis of dUTP to dUMP but with a low affinity for the substrate (K-m>100 muM). According to limited proteolysis, the C-terminal residues constitute a flexible region. The other protein investigated, MJ1102, is a specific dUTPase with a K-m for dUTP (0.4 muM) comparable in magnitude with that found for previously characterized dUTPases. Its physiological function is probably to degrade dUTP derived from other reactions in nucleotide metabolism.