Structure of human MTH1, a nudix family hydrolase that selectively degrades oxidized purine nucleoside triphosphates
Structure of human MTH1, a nudix family hydrolase that selectively degrades oxidized purine nucleoside triphosphates
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DOI：
10.1074/jbc.m402393200
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发表时间：
2004-08-06
影响因子：
4.8
通讯作者：
Shirakawa, M
中科院分区：
文献类型：
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作者：
Mishima, M;Sakai, Y;Shirakawa, M
通过正常细胞呼吸过程产生的氧自由基可导致基因组和线粒体DNA突变。人MTH 1将氧化嘌呤核苷三磷酸（如8-氧代-dGTP和2-羟基-dATP）水解为单磷酸，从而防止复制期间这些氧化核苷酸的错误掺入。在这里，我们提出的解决方案MTH 1的多维异构NMR光谱的结构。该蛋白采用了类似于大肠杆菌MutT的折叠，尽管这些蛋白质之间的低序列相似性以外的保守的Nutrium基序。从化学位移扰动实验中推导出的MTH 1的底物结合口袋位于与MutT基本相同的位置;然而，MTH 1中的口袋形成螺旋在很大程度上被置换（类似于9埃），使得口袋的形状在两种蛋白质之间不同。对形成口袋的残基的详细分析使我们能够将Asn（33）鉴定为MTH 1中区分嘌呤氧化形式的关键残基之一，并且该残基的突变改变了底物特异性。我们还表明，MTH 1催化水解的8-氧代-dGTP通过亲核取代水的β-磷酸。
Oxygen radicals generated through normal cellular respiration processes can cause mutations in genomic and mitochondrial DNA. Human MTH1 hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP and 2-hydroxy-dATP, to monophosphates, thereby preventing the misincorporation of these oxidized nucleotides during replication. Here we present the solution structure of MTH1 solved by multidimensional heteronuclear NMR spectroscopy. The protein adopts a fold similar to that of Escherichia coli MutT, despite the low sequence similarity between these proteins outside the conserved Nudix motif. The substrate-binding pocket of MTH1, deduced from chemical shift perturbation experiments, is located at essentially the same position as in MutT; however, a pocket-forming helix is largely displaced in MTH1 ( similar to 9 Angstrom) such that the shape of the pocket differs between the two proteins. Detailed analysis of the pocket-forming residues enabled us to identify Asn(33) as one of the key residues in MTH1 for discriminating the oxidized form of purine, and mutation of this residue modifies the substrate specificity. We also show that MTH1 catalyzes hydrolysis of 8-oxo-dGTP through nucleophilic substitution of water at the beta-phosphate.