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Fusion with anticodon binding domain of GluRS is not sufficient to alter the substrate specificity of a chimeric Glu-Q-RS
Published in Springer New York LLC
Volume: 33
Issue: 1
Pages: 48 - 60
Glutamyl-queuosine-tRNAAsp synthetase (Glu-Q-RS) is a paralog of glutamyl-tRNA synthetase (GluRS) and is found in more than forty species of proteobacteria, cyanobacteria, and actinobacteria. Glu-Q-RS shows striking structural similarity with N-terminal catalytic domain of GluRS (NGluRS) but it lacks the C-terminal anticodon binding domain (CGluRS). In spite of structural similarities, Glu-Q-RS and NGluRS differ in their functional properties. Glu-Q-RS glutamylates the Q34 nucleotide of the anticodon of tRNAAsp whereas NGluRS constitutes the catalytic domain of GluRS catalyzing the transfer of Glu on the acceptor end of tRNAGlu. Since NGluRS is able to catalyze aminoacylation of only tRNAGlu the glutamylation capacity of tRNAAsp by Glu-Q-RS is surprising. To understand the substrate specificity of Glu-Q-RS we undertook a systemic approach by investigating the biophysical and biochemical properties of the NGluRS (1-301), CGluRS (314-471) and Glu-Q-RS-CGluRS, (1-298 of Glu-Q-RS fused to 314-471 from GluRS). Circular dichroism, fluorescence spectroscopy and differential scanning calorimetry analyses revealed absence of N-terminal domain (1-298 of Glu-Q-RS) and C-terminal domain (314-471 from GluRS) communication in chimera, in contrast to the native full length GluRS. The chimeric Glu-Q-RS is still able to aminoacylate tRNAAsp but has also the capacity to bind tRNAGlu. However the chimeric protein is unable to aminoacylate tRNAGlu probably as a consequence of the lack of domain-domain communication. © Springer Science+Business Media New York 2013.
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