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Structural and molecular functional analysis of house-cleaning enzymes from an extreme thermophile

极端嗜热菌的房屋清洁酶的结构和分子功能分析

基本信息

批准号：
15570114
负责人：
MASUI Ryoji
金额：
$ 2.24万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570114/
关键词：
extreme thermophile Thermus thermophilus HB8 Nudix protein house-cleanin enzyme nucleotide metabolism structural analysis metal ion MuiT

项目摘要

Nudix proteins are hydrolases that degrade toxic nucleotides and excess nucleotide derivatives in cells and thus called "house-cleaning enzymes. " Substrates of these enzymes are nucleoside diphosphate linked to other moiety, X. Nudes proteins are involved in maintenance of genetic information and regulation of cell proliferation and signal transduction by degrading their substrates. This study aimed to elucidate substrate-recognition and catalytic mechanisms of nudix proteins (Ndx1-8) from an extreme thermophile Thermus thermophilus HB8 at an atomic level, by structural and molecular functional analysis. First, the crystal structures of the substrate-bound complex of Ndx1, an Ap6A hydrolase, were determined. Mutational analysis based on the resolved structures revealed that adenine and three phosphate moieties are important for substrate recognition and that two glutamate residues coordinated to divalent rations in the active site. Second, we determined seven structures of Ndx4, ADP-ribose pyrophosphatas (ADPRase), including that of the free enzyme, the Zn^<2+>-bound enzyme, the binary complex with ADP-ribose, the ternary complexes with ADP-ribose and Zn^<2+> or Gd^<3+>, and the product complexes with AMP and Mg^<2+> or with ribose-5'-phosphate and Zn^<2+>. We have prepared mutants based on its crystal structure and analyzed their activity. We have identified residues involved in the substrate binding and reveal a unique mode of substrate recognition displayed by the ADPRase group of enzymes. Furthermore, mutational and kinetic studies of residues conserved in the nudix motif have allowed us to propose a new catalytic model. This model differs from that proposed for other nudix proteins. The crystal structure of Ndx2 was also determined. Our study demonstrates the diversity of molecular mechanisms shown in the nudix family of proteins.

营养蛋白是降解细胞中有毒核苷酸和过量核苷酸衍生物的水解酶，因此被称为“房屋清洁酶”。“这些酶的底物是与其他部分X连接的核苷二磷酸。裸蛋白通过降解底物参与遗传信息的维持、细胞增殖和信号转导的调控。本研究旨在通过结构和分子功能分析，从原子水平上阐明极端嗜热菌Thermus thermophilus HB 8营养蛋白（Ndx 1 -8）的底物识别和催化机制。首先，测定了Ap 6A水解酶Ndx 1的底物结合复合物的晶体结构。基于解析结构的突变分析表明，腺嘌呤和三个磷酸部分是重要的底物识别和两个谷氨酸残基协调的活性位点中的二价口粮。其次，我们确定了Ndx 4，ADP-核糖焦磷酸酶（ADPRase）的七种结构，包括游离酶、Zn^<2+>结合酶、ADP-核糖二元复合物、ADP-核糖与Zn^<2+>或Gd^<3+>的三元复合物、AMP与Mg^<2+>或核糖-5 '-磷酸与Zn^<2+>的产物复合物。我们根据其晶体结构制备了突变体，并分析了它们的活性。我们已经确定了参与底物结合的残基，并揭示了ADPRase组酶显示的底物识别的独特模式。此外，突变和动力学的研究保守的nutrient基序的残基，使我们能够提出一个新的催化模型。该模型不同于其他营养素蛋白的模型。测定了Ndx 2的晶体结构。我们的研究证明了在营养素家族蛋白质中所显示的分子机制的多样性。