Fusion with Anticodon Binding Domain of GluRS is Not Sufficient to Alter the Substrate Specificity of a Chimeric Glu-Q-RS

Glutamyl-queuosine-tRNA^Asp 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 tRNA^Asp whereas NGluRS constitutes the catalytic domain of GluRS catalyzing the transfer of Glu on the acceptor end of tRNA^Glu. Since NGluRS is able to catalyze aminoacylation of only tRNA^Glu the glutamylation capacity of tRNA^Asp 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 tRNA^Asp but has also the capacity to bind tRNA^Glu. However the chimeric protein is unable to aminoacylate tRNA^Glu probably as a consequence of the lack of domain–domain communication.