Characterization of the vaccinia virus D10 decapping enzyme provides evidence for a two-metal-ion mechanism.
Decapping enzymes are required for the removal of the 5'-end cap of mRNAs. These enzymes exhibit a specific hydrolase activity, resulting in cleavage between the alpha- and beta-phosphates of the m7GpppN cap to generate both m7GDP and monophosphorylated RNA products. Decapping enzymes have been found in humans, plants and yeasts, and have been discovered more recently in vaccinia virus (D10 protein). Although experimental evidences are lacking, three-metal- and two-metal-ion mechanisms have been proposed so far for the decapping enzymes. In the present study, we performed a biochemical characterization of the interaction of bivalent cations with the vaccinia virus D10 protein. Synergistic activation of the enzyme was observed in the presence of Mg2+ and Mn2+ ions, suggesting the existence of two metal-ion-binding sites on the D10 protein. Moreover, dual-ligand titration experiments using fluorescence spectroscopy demonstrated the presence of two metal-ion-binding sites on the enzyme. A three-dimensional structural model of the active site of the enzyme was generated which highlighted the importance of three glutamate residues involved in the co-ordination of two metal ions and a water molecule. Mutational analyses confirmed the role of two glutamate residues for the binding of metal ions. We demonstrate that one metal ion is co-ordinated by Glu132, while the second metal ion is co-ordinated by Glu145. Taken together, these results support the proposed two-metal-ion mechanistic model for the D10 decapping enzyme.
Soulière
Marie F
MF
RNA Group/Groupe ARN, Département de Biochimie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada, J1H 5N4.
Perreault
Jean-Pierre
JP
Bisaillon
Martin
M
D001665:Binding Sites; D020134:Catalytic Domain; D002413:Cations, Divalent; D018698:Glutamic Acid; D008274:Magnesium; D008345:Manganese; D008670:Metals / Q000737:chemistry*; D009708:Nucleotidases / Q000737:chemistry*; D014616:Vaccinia virus / Q000201:enzymology*; D014764:Viral Proteins / Q000737:chemistry*
D002413:Cations, Divalent; D008670:Metals; D014764:Viral Proteins; D018698:Glutamic Acid; D008345:Manganese; C519568:D10 protein, vaccinia virus; D009708:Nucleotidases; D008274:Magnesium