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首页> 外文期刊>Journal of Molecular Biology >Structural basis for recognition and catalysis by the bifunctional dCTP deaminase and dUTPase from Methanococcus jannaschii.
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Structural basis for recognition and catalysis by the bifunctional dCTP deaminase and dUTPase from Methanococcus jannaschii.

机译：詹氏甲烷球菌的双功能dCTP脱氨酶和dUTPase识别和催化的结构基础。

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摘要

Potentially mutagenic uracil-containing nucleotide intermediates are generated by deamination of dCTP, either spontaneously or enzymatically as the first step in the conversion of dCTP to dTTP. dUTPases convert dUTP to dUMP, thus avoiding the misincorporation of dUTP into DNA and creating the substrate for the next enzyme in the dTTP synthetic pathway, thymidylate synthase. Although dCTP deaminase and dUTPase activities are usually found in separate but homologous enzymes, the hyperthermophile Methanococcus jannaschii has an enzyme, DCD-DUT, that harbors both dCTP deaminase and dUTP pyrophosphatase activities. DCD-DUT has highest activity on dCTP, followed by dUTP, and dTTP inhibits both the deaminase and pyrophosphatase activities. To help clarify structure-function relationships for DCD-DUT, we have determined the crystal structure of the wild-type DCD-DUT protein in its apo form to 1.42A and structures of DCD-DUT in complex with dCTP and dUTP to resolutions of 1.77A and 2.10A, respectively. To gain insights into substrate interactions, we complemented analyses of the experimentally defined weak density for nucleotides with automated docking experiments using dCTP, dUTP, and dTTP. DCD-DUT is a hexamer, unlike the homologous dUTPases, and its subunits contain several insertions and substitutions different from the dUTPase beta barrel core that likely contribute to dCTP specificity and deamination. These first structures of a dCTP deaminase reveal a probable role for an unstructured C-terminal region different from that of the dUTPases and possible mechanisms for both bifunctional enzyme activity and feedback inhibition by dTTP.
机译：作为dCTP转化为dTTP的第一步，通过自发或酶促脱氨处理dCTP来产生潜在的诱变的含尿嘧啶核苷酸中间体。 dUTPases将dUTP转换为dUMP，从而避免了dUTP误掺入DNA，并为dTTP合成途径中的下一个酶胸苷酸合酶创造了底物。尽管通常在单独但同源的酶中发现dCTP脱氨酶和dUTPase活性，但超嗜热菌詹氏甲烷球菌具有一种DCD-DUT酶，既具有dCTP脱氨酶又具有dUTP焦磷酸酶活性。 DCD-DUT对dCTP的活性最高，其次是dUTP，而dTTP抑制脱氨酶和焦磷酸酶的活性。为了帮助阐明DCD-DUT的结构-功能关系，我们确定了apo形式的野生型DCD-DUT蛋白的晶体结构为1.42A，与dCTP和dUTP配合使用的DCD-DUT结构的分辨率为1.77。 A和2.10A。为了深入了解底物相互作用，我们使用自动对接实验（使用dCTP，dUTP和dTTP）对核苷酸的实验定义的弱密度分析进行了补充。 DCD-DUT是六聚体，与同源的dUTPases不同，它的亚基包含几个不同于dUTPaseβ桶形核心的插入和取代，可能有助于dCTP特异性和脱氨作用。 dCTP脱氨酶的这些第一个结构显示了与dUTPases不同的非结构化C末端区域的可能作用，以及双功能酶活性和dTTP抑制反馈的可能机制。

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