Structure-based mutational and functional analysis identify human NM23-H2 as a multifunctional enzyme
Structure-based mutational and functional analysis identify human NM23-H2 as a multifunctional enzyme
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DOI：
10.1021/bi025606
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发表时间：
2002-05-21
期刊：
影响因子：
2.9
通讯作者：
Xu, YW
中科院分区：
文献类型：
--
作者：
Postel, EH;Abramczyk, BA;Xu, YW
人NM 23-H2蛋白是一种转录调节因子（PuF），通过共价键形成结合和切割DNA，还催化磷酰基转移（NDP激酶）。我们以前的工作已经确定了两个独立的DNA结合，区域对NM 23-H2/PUF：一个序列依赖的DNA结合表面，涉及残基Ar 34，Asn 69，和Lys 134的赤道上的六聚体蛋白和一个共价的DNA结合位点，涉及Lys 12位于核苷酸结合位点，NDP激酶反应的网站。为了了解核苷酸结合位点在DNA切割反应中的作用，并建立核酸酶和NDP激酶活性之间的联系，我们使用与GDP复合的NM 23-H2的已知晶体结构作为定点诱变的基础。因此，我们确定Arg 88和Arg 105作为残基，除了Lys 12，关键的共价DNA结合和DNA切割，以及NDP激酶反应。另一个残基，Gln 17，只需要DNA切割，和Tyr 52，Asn 115，和His 118被发现是必不可少的，只有NDP激酶活性。与核苷酸结合位点相关的这七个功能上重要的氨基酸中的六个在进化上是保守的，强调了它们的生物学重要性。我们还表明，核苷三磷酸，但不核苷二磷酸抑制共价DNA结合和DNA裂解反应，独立的磷酰基转移和NDP激酶反应。这些发现共同表明，单核苷酸和双链DNA寡核苷酸在核苷酸结合位点的结合模式不同，并且NM 23-H2具有多种生物化学活性。与这些意见一致的模型。
The human NM23-H2 protein is a transcriptional regulator (PuF) that binds and cleaves DNA via covalent bond formation, and also catalyzes phosphoryl transfer (NDP kinase). Our previous work has identified two separate DNA-binding, regions on NM23-H2/PuF: a sequence-dependent DNA-binding surface involving, residues Ar34, Asn69, and Lys134 on the equator of the hexameric protein and a covalent DNA-binding site involving Lys12 located in the nucleotide-binding site, the site of the NDP kinase reaction. To understand the role of the nucleotide-binding site in the DNA cleavage reaction and to establish a connection between the nuclease and the NDP kinase activities, we used the known crystal structure of NM23-H2 complexed with GDP as the basis for site-directed mutagenesis. We thus identified Arg88 and Arg105 as residues that are, in addition to Lys12, critical for covalent DNA binding and DNA cleavage, as well as for the NDP kinase reaction. Another residue, Gln17, was required only for DNA cleavage, and Tyr52, Asn115, and His118 were found to be essential only for the NDP kinase activity. Six of these seven functionally important amino acids associated with the nucleotide-binding site are evolutionarily conserved, underscoring their biological importance. We also show that nucleoside triphosphates but not nucleoside diphosphates inhibited the covalent DNA binding and DNA cleavage reactions, independent of phosphoryl transfer and the NDP kinase reaction. These findings collectively suggest that the binding modes of mononucleotides and duplex DNA oligonucleotides in the nucleotide-binding site differ, and that NM23-H2 possesses multiple biochemical activities. A model consistent with these observations is presented.