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.

Nudix蛋白是水解酶，降解细胞中毒性毒性核苷酸和过量的核苷酸衍生物，因此称为“家庭清洁酶”。这些酶的底物是与其他部分相关的核苷二磷酸，X。Nudes蛋白参与维持遗传信息和调节的调节。细胞增殖和信号转导通过降解其底物。这项研究旨在通过结构和分子功能分析从原子水平阐明nudix蛋白（NDX1-8）的Nudix蛋白（NDX1-8）的底物识别和催化机制。首先，确定了NDX1的基质结合复合物的晶体结构，即AP6A水解酶。基于解析结构的突变分析表明，腺嘌呤和三个磷酸盐部分对于底物识别很重要，并且两个谷氨酸残基与活性部位的二价配给协调。其次，我们确定了NDX4，ADP-核糖焦磷酸（Adprase）的七个结构，包括游离酶的Zn^<2+> - 结合酶，带有ADP-核糖的二进制复合物，带有ADP-核糖的二元络合物，和Zn^<2+>或gd^<3+>，以及用AMP和Mg^<2+>或核糖-5'-磷酸盐和Zn^<2+>的产品配合物。我们已经根据突变体的晶体结构制备，并分析了它们的活性。我们已经确定了涉及底物结合的残基，并揭示了Adprase酶组显示的独特底物识别模式。此外，在Nudix基序中保守的残基的突变和动力学研究使我们能够提出一种新的催化模型。该模型与针对其他Nudix蛋白的建议不同。还确定了NDX2的晶体结构。我们的研究证明了Nudix蛋白质家族中显示的分子机制的多样性。