Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poly(A)-specific ribonuclease (PARN) is a highly poly(A)-specific 3'-exoribonuclease that efficiently degrades mRNA poly(A) tails. PARN belongs to the DEDD family of nucleases, and four conserved residues are essential for PARN activity, i.e. Asp-28, Glu-30, Asp-292, and Asp-382. Here we have investigated how catalytically important divalent metal ions are coordinated in the active site of PARN. Each of the conserved amino acid residues was substituted with cysteines, and it was found that all four mutants were inactive in the presence of Mg2+. However, in the presence of Mn2+, Zn2+, Co2+, or Cd2+, PARN activity was rescued from the PARN(D28C), PARN(D292C), and PARN(D382C) variants, suggesting that these three amino acids interact with catalytically essential metal ions. It was found that the shortest sufficient substrate for PARN activity was adenosine trinucleotide (A3) in the presence of Mg2+ or Cd2+. Interestingly, adenosine dinucleotide (A) was efficiently hydrolyzed in the presence of Mn2+, Zn2+, or Co2+, suggesting that the substrate length requirement for PARN can be modulated by the identity of the divalent metal ion. Finally, introduction of phosphorothioate modifications into the A substrate demonstrated that the scissile bond non-bridging phosphate oxygen in the pro-R position plays an important role during cleavage, most likely by coordinating a catalytically important divalent metal ion. Based on our data we discuss binding and coordination of divalent metal ions in the active site of PARN.
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PMID:Coordination of divalent metal ions in the active site of poly(A)-specific ribonuclease. 1535 88

Poly(A)-specific ribonuclease (PARN), a member of the DEDD family, is a key enzyme involved in the deadenylation of mRNA in higher eukaryotic cells. In this research, it was found that Mg(2+) could protect PARN against thermal inactivation by increasing the midpoint of inactivation and decreasing the inactivation rate. This protective effect was unique to Mg(2+) in a concentration-dependent manner. However, the thermal unfolding and aggregation was promoted by the addition of Mg(2+) at high temperatures. These results revealed that Mg(2+) might have dual effects on PARN stability: protecting the active site but endangering the overall structural stability.
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PMID:Effect of magnesium ions on the thermal stability of human poly(A)-specific ribonuclease. 1730 97

The nsp14 protein, an exoribonuclease of the DEDD superfamily encoded by severe acute respiratory syndrome coronavirus (SARS-CoV), was expressed in fusion with different affinity tags. The recombinant nspl4 proteins with either GST fusion or 6-histidine tag were shown to possess ribonuclease activity but nspl4 with a short MGHHHHHHGS tag sequence at the N-terminus increased the solubility of nspl4 protein and facilitated the protein purification. Mutations of the conserved residues of nspl4 resulted in significant attenuation but not abolishment of the ribonuclease activity. Combination of fluorescence and circular dichroism spectroscopy analyses showed that the conformational stability of nsp14 protein varied with many external factors such as pH, temperature and presence of denaturing chemicals. These results provide new information on the structural features and would be helpful for further characterization of this functionally important protein.
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PMID:[Synthesis in Escherichia coli cells and characterization of the active exoribonuclease of severe acute respiratory syndrome coronavirus]. 1954 31

Divalent metal ions are essential for the efficient catalysis and structural stability of many nucleotidyl-transfer enzymes. Poly(A)-specific ribonuclease (PARN) belongs to the DEDD superfamily of 3'-exonucleases, and the active site of PARN contains four conserved acidic amino acid residues that coordinate two Mg(2+) ions. In this research, we studied the roles of these four acidic residues in PARN thermal stability by mutational analysis. It was found that Mg(2+) significantly decreased the rate but increased the aggregate size of the 54 kDa wild-type PARN in a concentration-dependent manner. All of the four mutants decreased PARN thermal aggregation, while the aggregation kinetics of the mutants exhibited dissimilar Mg(2+)-dependent behavior. A comparison of the kinetic parameters indicated that Asp28 was the most crucial one to the binding of the two Mg(2+) ions, while metal B might be more important in PARN structural stability. The spectroscopic and aggregation results also suggested that the alterations in the active site structure by metal binding or mutations might lead to a global conformational change of the PARN molecule.
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PMID:Dissimilar roles of the four conserved acidic residues in the thermal stability of poly(A)-specific ribonuclease. 2168 57

In eukaryotic cells, the shortening and removal of the poly(A) tail of cytoplasmic mRNA by deadenylase enzymes is a critical step in post-transcriptional gene regulation. The ribonuclease activity of deadenylase enzymes is attributed to either a DEDD (Asp-Glu-Asp-Asp) or an endonuclease-exonuclease-phosphatase domain. Both domains require the presence of two Mg2+ ions in the active site. To facilitate the biochemical analysis of deadenylase enzymes, we have developed a fluorescence-based deadenylase assay. The assay is based on end-point measurement, suitable for quantitative analysis and can be adapted for 96- and 384-well microplate formats. We demonstrate the utility of the assay by screening a chemical compound library, resulting in the identification of non-nucleoside inhibitors of the Caf1/CNOT7 enzyme, a catalytic subunit of the Ccr4-Not deadenylase complex. These compounds may be useful tools for the biochemical analysis of the Caf1/CNOT7 deadenylase subunit of the Ccr4-Not complex and indicate the feasibility of developing selective inhibitors of deadenylase enzymes using the fluorescence-based assay.
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PMID:A fluorescence-based assay suitable for quantitative analysis of deadenylase enzyme activity. 2417 Aug 10

The shortening of the poly(A) tail of cytoplasmic mRNA (deadenylation) is a pivotal step in the regulation of gene expression in eukaryotic cells. Deadenylation impacts on both regulated mRNA decay as well as the rate of mRNA translation. An important enzyme complex involved in poly(A) shortening is the Ccr4-Not deadenylase. In addition to at least six non-catalytic subunits, it contains two distinct subunits with ribonuclease activity: a Caf1 subunit, characterized by a DEDD (Asp-Glu-Asp-Asp) domain, and a Ccr4 component containing an endonuclease-exonuclease-phosphatase (EEP) domain. In vertebrate cells, the complexity of the complex is further increased by the presence of paralogs of the Caf1 subunit (encoded by either CNOT7 or CNOT8) and the occurrence of two Ccr4 paralogs (encoded by CNOT6 or CNOT6L). In plants, there are also multiple Caf1 and Ccr4 paralogs. Thus, the composition of the Ccr4-Not complex is heterogeneous. The potential differences in the intrinsic enzymatic activities of the paralogs will be discussed. In addition, the potential redundancy, cooperation, and/or the extent of unique roles for the deadenylase subunits of the Ccr4-Not complex will be reviewed. Finally, novel approaches to study the catalytic roles of the Caf1 and Ccr4 subunits will be discussed.
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PMID:Heterogeneity and complexity within the nuclease module of the Ccr4-Not complex. 2439 63

The nsp14 protein, an exoribonuclease of the DEDD superfamily encoded by severe acute respiratory syndrome coronavirus (SARS-CoV), was expressed in fusion with different affinity tags. The recombinant nsp14 proteins with either GST fusion or 6-histidine tag were shown to possess ribonuclease activity but nsp14 with a short MGHHHHHHGS tag sequence at the N-terminus increased the solubility of nsp14 protein and facilitated the protein purification. Mutations of the conserved residues of nsp14 resulted in significant attenuation but not abolishment of the ribonuclease activity. Combination of fluorescence and circular dichroism spectroscopy analyses showed that the conformational stability of nsp14 protein varied with many external factors such as pH, temperature and presence of denaturing chemicals. These results provide new information on the structural features and would be helpful for further characterization of this functionally important protein.
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PMID:Synthesis in Escherichia coli cells and characterization of the active exoribonuclease of severe acute respiratory syndrome coronavirus. 3221 68