Crystal Structures of the Novel Cytosolic 5′-Nucleotidase IIIB Explain Its Preference for m7GMP
Crystal Structures of the Novel Cytosolic 5′-Nucleotidase IIIB Explain Its Preference for m7GMP
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DOI：
10.1371/journal.pone.0090915
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发表时间：
2014-03-06
期刊：
影响因子：
3.7
通讯作者：
Ficner, Ralf
中科院分区：
文献类型：
--
作者：
Monecke, Thomas;Buschmann, Juliane;Ficner, Ralf
5'-核苷酸酶催化核苷单磷酸的水解去磷酸化。作为分解代谢酶，它们对细胞核苷酸水平的调节有显着贡献。核苷酸代谢失调和核苷酸酶缺乏与许多疾病有关。七种人类 5'-核苷酸酶在底物特异性和细胞定位方面有所不同。最近，新型胞质 5'-核苷酸酶 III 样蛋白（cN-IIIB）在人类和果蝇中得到了表征。 cN-IIIB 对修饰的核苷酸 7-甲基鸟苷单磷酸表现出强烈的底物偏好，但这种偏好的结构原因尚不清楚。在这里，我们展示了来自果蝇的 cN-IIIB 与反应产物 7-甲基鸟苷或胞苷结合的晶体结构。结构数据表明，产物的胞嘧啶和 7-甲基鸟嘌呤部分以共面但偏心的位置堆叠在两个芳香族残基之间。 7-甲基鸟苷通过 p-p 相互作用特异性结合，并通过芳香族侧链和带正电荷的 7-甲基鸟嘌呤之间额外的阳离子-p 库仑相互作用与未修饰的鸟苷区分开来。值得注意的是，通过垂直于嘌呤环的额外色氨酸堆积的 T 形边对面堆叠并与其他芳香物一起形成芳香槽，进一步稳定了碱基。结构数据与定点诱变实验相结合，揭示了 cN-IIIB 广泛底物特异性的分子基础，同时也解释了 7-甲基鸟苷单磷酸的底物偏好。通过诱变研究分析cN-IIIB和主要嘧啶59-核苷酸酶cN-IIIA的底物特异性，我们发现cN-IIIA也使嘌呤m(7)GMP去磷酸化，从而重新定义了其底物谱。与 cN-IIIA 和 m(7)GMP 的对接计算以及生化数据表明，Asn69 通常不排除嘌呤底物的周转，从而纠正了之前的建议。
5'-nucleotidases catalyze the hydrolytic dephosphorylation of nucleoside monophosphates. As catabolic enzymes they contribute significantly to the regulation of cellular nucleotide levels; misregulation of nucleotide metabolism and nucleotidase deficiencies are associated with a number of diseases. The seven human 5'-nucleotidases differ with respect to substrate specificity and cellular localization. Recently, the novel cytosolic 5'-nucleotidase III-like protein, or cN-IIIB, has been characterized in human and Drosophila. cN-IIIB exhibits a strong substrate preference for the modified nucleotide 7-methylguanosine monophosphate but the structural reason for this preference was unknown. Here, we present crystal structures of cN-IIIB from Drosophila melanogaster bound to the reaction products 7-methylguanosine or cytidine. The structural data reveal that the cytosine-and 7-methylguanine moieties of the products are stacked between two aromatic residues in a coplanar but off-centered position. 7-methylguanosine is specifically bound through p-p interactions and distinguished from unmodified guanosine by additional cation-p coulomb interactions between the aromatic side chains and the positively charged 7-methylguanine. Notably, the base is further stabilized by T-shaped edge-to-face stacking of an additional tryptophan packing perpendicularly against the purine ring and forming, together with the other aromates, an aromatic slot. The structural data in combination with site-directed mutagenesis experiments reveal the molecular basis for the broad substrate specificity of cN-IIIB but also explain the substrate preference for 7-methylguanosine monophosphate. Analyzing the substrate specificities of cN-IIIB and the main pyrimidine 59-nucleotidase cN-IIIA by mutagenesis studies, we show that cN-IIIA dephosphorylates the purine m(7)GMP as well, hence redefining its substrate spectrum. Docking calculations with cN-IIIA and m(7)GMP as well as biochemical data reveal that Asn69 does not generally exclude the turnover of purine substrates thus correcting previous suggestions.