Structural and functional characterization of mitochondrial EndoG, a sugar non-specific nuclease which plays an important role during apoptosis
Structural and functional characterization of mitochondrial EndoG, a sugar non-specific nuclease which plays an important role during apoptosis
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DOI：
10.1016/j.jmb.2004.02.069
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发表时间：
2004-04-23
影响因子：
5.6
通讯作者：
Meiss, G
中科院分区：
文献类型：
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作者：
Schäfer, P;Scholz, SR;Meiss, G
结合序列分析，结构预测，和定点突变，我们已经研究了催化和底物结合的凋亡线粒体核酸酶EndoG，这属于大家庭的DNA/RNA非特异性betabetaalpha-Me-finger核酸酶的机制。磷酸二酯键断裂的催化作用涉及几个高度保守的氨基酸残基，即水活化和金属离子结合所需的His 143、Asn 174和Glu 182，以及适当底物结合和定位所需的Arg 141。这些结果表明，EndoG基本上遵循与沙雷氏菌核酸酶类似的机制，沙雷氏菌核酸酶是DNA/RNA非特异性核酸酶家族的最佳研究代表，但观察到过渡态稳定化的差异。此外，我们已经确定了两个推定的DNA/RNA结合残基的牛EndoG，Arg 135和Arg 186，严格保守的核酸酶家族的哺乳动物成员之间，这表明一个类似的模式结合到单链和双链核酸底物的这些酶。最后，我们证明了异位表达的活性和非活性变体的牛EndoG在HeLa和CV1-细胞，extramitriotalactive EndoG本身诱导细胞死亡，而表达的酶活性的变体不。(C)2004爱思唯尔有限公司保留所有权利。
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. (C) 2004 Elsevier Ltd. All rights reserved.