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

Nudix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”。这些酶的底物是核苷二磷酸与其他部分连接，X. Nudes蛋白通过降解底物参与维持遗传信息和调节细胞增殖和信号转导。本研究旨在通过结构和分子功能分析，在原子水平上阐明极端嗜热菌HB8中nudix蛋白（Ndx1-8）的底物识别和催化机制。首先，测定了Ap6A水解酶Ndx1的底物结合配合物的晶体结构。基于分解结构的突变分析表明，腺嘌呤和三个磷酸基团对底物识别很重要，两个谷氨酸残基在活性位点协调为二价定量。其次，我们确定了Ndx4 adp -核糖焦磷酸酶（ADPRase）的7种结构，包括游离酶、Zn^<2+>结合酶、adp -核糖的二元配合物、adp -核糖与Zn^<2+>或Gd^<3+>的三元配合物、AMP与Mg^<2+>或5'-磷酸核糖与Zn^<2+>的产物配合物。根据其晶体结构制备了突变体，并对其活性进行了分析。我们已经确定了参与底物结合的残基，并揭示了ADPRase酶组所显示的一种独特的底物识别模式。此外，对nudix基序中保守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。该模型不同于其他裸蛋白的模型。测定了Ndx2的晶体结构。我们的研究显示了nudix蛋白家族分子机制的多样性。

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.