Gln-tRNA^Gln formation from Glu-tRNA^Gln requires cooperation of an asparaginase and a Glu-tRNA^Gln kinase

Gln-tRNA^Gln is synthesized from Glu-tRNA^Gln in most microorganisms by a tRNA-dependent amidotransferase in a reaction requiring ATP and an amide donor such as glutamine. GatDE is a heterodimeric amidotransferase that is ubiquitous in Archaea. GatD resembles bacterial asparaginases and is expected to function in amide donor hydrolysis. We show here that Methanothermobacter thermautotrophicus GatD acts as a glutaminase but only in the presence of both Glu-tRNA^Gln and the other subunit, GatE. The fact that only Glu-tRNA^Gln but not tRNA^Gln could activate the glutaminase activity of GatD suggests that glutamine hydrolysis is coupled tightly to transamidation. M. thermautotrophicus GatDE enzymes that were mutated in GatD at each of the four critical asparaginase-active site residues lost the ability to hydrolyze glutamine and were unable to convert Glu-tRNA ^Gln to Gln-tRNA^Gln when glutamine was the amide donor. However, ammonium chloride rescued the activities of these mutants, suggesting that the integrity of the ATPase and the transferase activities in the mutant GatDE enzymes was maintained. In addition, pyroglutamyl-tRNA^Gln accumulated during the reaction catalyzed by the glutaminase-deficient mutants or by GatE alone. The pyroglutamyl-tRNA is most likely a cyclized by-product derived from γ-phosphoryl-Glu-tRNA^Gln, the proposed high energy intermediate in Glu-tRNA^Gln transamidation. That GatE alone could form the intermediate indicates that GatE is a Glu-tRNA^Gln kinase. The activation of Glu-tRNA^Gln via γ-phosphorylation bears a similarity to the mechanism used by glutamine synthetase, which may point to an ancient link between glutamine synthesized for metabolism and translation.