The crystal structure and mutational analysis of human NUDT9
The crystal structure and mutational analysis of human NUDT9
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DOI：
10.1016/s0022-2836(03)00954-9
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发表时间：
2003-09-12
影响因子：
5.6
通讯作者：
Stoddard, BL
中科院分区：
文献类型：
--
作者：
Shen, BW;Perraud, AL;Stoddard, BL
人ADP-核糖焦磷酸酶NUDT 9属于在细胞中分解代谢潜在毒性化合物的Nutrius水解酶的超家族。该酶水解ADP-核糖（ADPR）为AMP和核糖5 '-磷酸。NUDT 9与ADPR门控钙通道TRPM 2的C末端胞质结构域具有39%的序列同一性，TRPM 2表现出低但特异的酶活性。我们确定了NUDT 9在反应产物核糖5-磷酸存在和不存在下的晶体结构。在此基础上，结构和与细菌同源物的比较，建立了底物复合物的模型。测定了模拟离子通道结构域的NuFlo标签的双点突变体（R229 E230 F231至R229 I230 L231）的结构和活性。最后，将一对另外的突变构建体的活性与野生型酶进行比较。第一个对应于一个最小的Nutrient域缺少一个N-末端域和C-末端尾;第二个破坏了活性位点中的两个潜在的通用碱基。NUDT 9含有一个具有新折叠的N-末端结构域和一个催化性C-末端NuDT结构域。与其最接近的功能同源物（同源二聚体大肠杆菌ADPRase）不同，它作为单体具有活性，底物结合在结构域之间的裂缝中。RIL突变体的结构为TRPM 2离子通道活性降低提供了结构基础。ADPR底物的构象和结合作用与E. coli ADPRase;在它们的活性位点上结构对齐的酸性残基的突变对催化效率产生显著不同的影响，表明它们的反应途径和机制可能已经分化。(C)2003爱思唯尔有限公司。保留所有权利。
Human ADP-ribose pyrophosphatase NUDT9 belongs to a superfamily of Nudix hydrolases that catabolize potentially toxic compounds in the cell. The enzyme hydrolyzes ADP-ribose (ADPR) to AMP and ribose 5'-phosphate. NUDT9 shares 39% sequence identity with the C-terminal cytoplasmic domain of the ADPR-gated calcium channel TRPM2, which exhibits low but specific enzyme activity. We determined crystal structures of NUDT9 in the presence and in the absence of the reaction product ribose 5-phosphate. On the basis of these, structures and comparison with a bacterial homologue, a model of the substrate complex was built. The structure and activity of a double point mutant (R229E230F231 to R229I230L231),which mimics the Nudix signature of the ion channel domain, was determined. Finally, the activities of a pair of additional mutated constructs were compared to the wild-type enzyme. The first corresponds to a minimal Nudix domain missing an N-terminal domain and C-terminal tail; the second disrupts two potential general bases in the active site. NUDT9 contains an N-terminal domain with a novel fold and a catalytic C-terminal Nudix domain. Unlike its closest functional homologue (homodimeric Escherichia coli ADPRase), it is active as a monomer, and the substrate is bound in a cleft between the domains. The structure of the RIL mutant provides structural basis for the reduced activity of the TRPM2 ion channel. The conformation and binding interactions of ADPR substrate are predicted to differ from those observed for E. coli ADPRase; mutation of structurally aligned acidic residues in their active sites produce significantly different effects on catalytic efficiency, indicating that their reaction pathways and mechanisms may have diverged. (C) 2003 Elsevier Ltd. All rights reserved.