首页> 外文期刊>Journal of Molecular Biology >Structural and Functional Characterization of Mitochondrial EndoG, a Sugar Non-specific Nuclease which Plays an Important Role During Apoptosis.
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Structural and Functional Characterization of Mitochondrial EndoG, a Sugar Non-specific Nuclease which Plays an Important Role During Apoptosis.

机译:线粒体End​​oG的结构和功能表征,一种糖非特异性核酸酶,在凋亡过程中起重要作用。

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Combining sequence analysis, structure prediction, and site-directed mutagenesis, we have investigated the mechanism of catalysis and substrate binding by the apoptotic mitochondrial nuclease EndoG, which belongs to the large family of DNA/RNA non-specific betabetaalpha-Me-finger nucleases. Catalysis of phosphodiester bond cleavage involves several highly conserved amino acid residues, namely His143, Asn174, and Glu182 required for water activation and metal ion binding, as well as Arg141 required for proper substrate binding and positioning, respectively. These results indicate that EndoG basically follows a similar mechanism as the Serratia nuclease, the best studied representative of the family of DNA/RNA non-specific nucleases, but that differences are observed for transition state stabilisation. In addition, we have identified two putative DNA/RNA binding residues of bovine EndoG, Arg135 and Arg186, strictly conserved only among mammalian members of the nuclease family, suggesting a similar mode of binding to single and double-stranded nucleic acid substrates by these enzymes. Finally, we demonstrate by ectopic expression of active and inactive variants of bovine EndoG in HeLa and CV1-cells that extramitochondrial active EndoG by itself induces cell death, whereas expression of an enzymatically inactive variant does not.
机译:结合序列分析,结构预测和定点诱变,我们研究了凋亡的线粒体核酸酶EndoG的催化和底物结合的机制,后者属于DNA / RNA非特异性βbetaalpha-Me-手指核酸酶的大家族。磷酸二酯键裂解的催化涉及几个高度保守的氨基酸残基,分别是水活化和金属离子结合所需的His143,Asn174和Glu182,以及适当的底物结合和定位所需的Arg141。这些结果表明,EndoG基本上遵循与Serratia核酸酶类似的机制,Serratia核酸酶是DNA / RNA非特异性核酸酶家族研究最好的代表,但观察到过渡态稳定的差异。此外,我们已经鉴定出牛EndoG的两个推定的DNA / RNA结合残基,Arg135和Arg186,仅在核酸酶家族的哺乳动物成员中严格保守,这表明这些酶与单链和双链核酸底物的结合方式相似。最后,我们通过在HeLa和CV1细胞中异位表达牛EndoG的活性和非活性变体来证明,线粒体活性EndoG本身会诱导细胞死亡,而酶无活性的变体则不会。

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