Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase -: Kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme
Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase -: Kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme
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DOI：
10.1074/jbc.m313643200
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发表时间：
2004-04-23
影响因子：
4.8
通讯作者：
Vértessy, BG
中科院分区：
文献类型：
--
作者：
Kovári, J;Barabás, O;Vértessy, BG
dUTPase通过排除尿嘧啶负责预防性DNA修复。果蝇酶的发育调控被认为与无胸腺嘧啶细胞凋亡有关。在这里，我们发现除了保守的dUTPase序列基序外，果蝇酶基因还编码一个独特的富含ala - pro的片段。重组蛋白和截断突变体的动力学和结构分析表明，Ala-Pro片段具有灵活性，在体外没有调节作用。同型三聚体酶以三聚体形式可逆展开，其熔融温度为54℃，比大肠杆菌dUTPase低23℃。与细菌酶相比，Mg2+结合调节了苍蝇dUTPase的构象，这是通过光谱和熔融温度的增加来确定的。通过有限胰蛋白酶分解的三个不同步骤，产生了一个折叠良好但无活性的同型三聚体核心结构域。在苍蝇中，而不是在细菌dUTPase中，产物dUMP的结合在胰蛋白酶位点诱导了对蛋白质水解的保护，反映了催化能力强的封闭构象的形成。结晶学分析表明，在结晶相中存在一种稳定的果蝇dUTPase单体。真核和原核dutpase的原型在活性位点的构象柔韧性、底物特异性、金属离子结合和晶体相的寡聚化方面存在显著差异，这与催化机制和亚基界面亲水性的改变是一致的。
dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54degreesC, 23degreesC lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg2+ binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.