Glu-tRNA<sup>Gln</sup> amidotransferase or glutaminyl-tRNA synthase (glutamine-hydrolysing) enzyme () is an amidotransferase that catalyzes the conversion of the non-cognate amino acid glutamyl-tRNA<sup>Gln</sup> to the cognate glutaminyl-tRNA<sup>Gln.</sup>. It catalyzes the reaction:
This enzyme belongs to the family of ligases, specifically those forming carbon-nitrogen bonds carbon-nitrogen ligases with glutamine as amido-N-donor. The systematic name of this enzyme class is glutamyl-tRNA<sup>Gln</sup>:L-glutamine amido-ligase (ADP-forming). This enzyme participates in glutamate metabolism and alanine and aspartate metabolism.
Most bacterial and all archaea genomes do not encode a glutaminyl-tRNA synthetase (GlnRS). Instead they first synthesize the attachment of an amino acid on the tRNA<sup>Gln</sup> by first attaching a non-cognate glutamate to the tRNA. Then these organisms use the amidotransferase: glutaminyl-tRNA synthase (glutamine-hydrolysing) () enzyme to convert the glutamate attached to tRNA<sup>Gln</sup> to glutamine.