Biochemical characterization of exoribonuclease encoded by SARS coronavirus.
The nsp14 protein is an exoribonuclease that is encoded by severe acute respiratory syndrome coronavirus (SARS-CoV). We have cloned and expressed the nsp14 protein in Escherichia coli, and characterized the nature and the role(s) of the metal ions in the reaction chemistry. The purified recombinant nsp14 protein digested a 5'-labeled RNA molecule, but failed to digest the RNA substrate that is modified with fluorescein group at the 3'-hydroxyl group, suggesting a 3'-to-5' exoribonuclease activity. The exoribonuclease activity requires Mg2+ as a cofactor. Isothermal titration calorimetry (ITC) analysis indicated a two-metal binding mode for divalent cations by nsp14. Endogenous tryptophan fluorescence and circular dichroism (CD) spectra measurements showed that there was a structural change of nsp14 when binding with metal ions. We propose that the conformational change induced by metal ions may be a prerequisite for catalytic activity by correctly positioning the side chains of the residues located in the active site of the enzyme.
Chen
Ping
P
State Key Laboratory of Virology and The Modern Virology Research Centre, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Jiang
Miao
M
Hu
Tao
T
Liu
Qingzhen
Q
Chen
Xiaojiang S
XS
Guo
Deyin
D
D001665:Binding Sites; D002151:Calorimetry; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D005095:Exoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology*; D013379:Substrate Specificity; D013816:Thermodynamics; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D002413:Cations, Divalent; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D008345:Manganese; C525596:nsp14 protein, SARS coronavirus; D005095:Exoribonucleases; D008274:Magnesium; D015032:Zinc