Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme.
Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme.
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DOI：
10.1042/bj20110304
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发表时间：
2011-07-15
期刊：
影响因子：
--
通讯作者：
Yakunin AF
中科院分区：
文献类型：
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作者：
Tchigvintsev A;Singer AU;Flick R;Petit P;Brown G;Evdokimova E;Savchenko A;Yakunin AF
所有自由生物的基因组都编码dUTPase(dUTP焦磷酸酶)，它在阻止尿嘧啶掺入DNA中起着关键作用。本文描述了酿酒酵母dUTPase(DUT1)的生化和结构特征。DUT1对dUTP的水解完全依赖于二价金属阳离子，在有Mg2+、Co2+、Mn2+、Ni2+或Zn2+存在的情况下，DUT1对dUTP的催化活性很高。此外，DUT1对另一种潜在的突变核苷酸：dITP显示出显著的活性。在这两种底物上，DUT1呈现出S形饱和曲线，表明亚基之间存在正的协同作用。DUT1的晶体结构是在非水解性底物α，β-imido dUTP或Dump产物的络合物作用下，在apo状态下以2？分辨率(1？=0.1 nm)解析得到的。活性中心残基的丙氨酸置换突变揭示了7个对活性重要的残基，其中包括保守的三联体Asp87/Arg137/Asp85。Y88A突变蛋白对dUTP和UTP具有同等的活性，表明这种保守的酪氨酸残基负责对核苷酸的歧视。DUT1的结构和定点突变支持保守的Phe142在与尿嘧啶碱基相互作用中的作用。我们的工作进一步揭示了dUTPase底物选择性和催化的分子机制。
Genomes of all free-living organisms encode the enzyme dUTPase (dUTP pyrophosphatase), which plays a key role in preventing uracil incorporation into DNA. In the present paper, we describe the biochemical and structural characterization of DUT1 (Saccharomyces cerevisiae dUTPase). The hydrolysis of dUTP by DUT1 was strictly dependent on a bivalent metal cation with significant activity observed in the presence of Mg2+, Co2+, Mn2+, Ni2+ or Zn2+. In addition, DUT1 showed a significant activity against another potentially mutagenic nucleotide: dITP. With both substrates, DUT1 demonstrated a sigmoidal saturation curve, suggesting a positive co-operativity between the subunits. The crystal structure of DUT1 was solved at 2 Å resolution (1 Å = 0.1 nm) in an apo state and in complex with the non-hydrolysable substrate α,β-imido dUTP or dUMP product. Alanine-replacement mutagenesis of the active-site residues revealed seven residues important for activity including the conserved triad Asp87/Arg137/Asp85. The Y88A mutant protein was equally active against both dUTP and UTP, indicating that this conserved tyrosine residue is responsible for discrimination against ribonucleotides. The structure of DUT1 and site-directed mutagenesis support a role of the conserved Phe142 in the interaction with the uracil base. Our work provides further insight into the molecular mechanisms of substrate selectivity and catalysis of dUTPases.