Gln-tRNAGln Formation from Glu-tRNAGln Requires Cooperation of an Asparaginase and a Glu-tRNAGln Kinase

Liang Feng,Kelly Sheppard,Debra Tumbula-Hansen,Dieter Söll,Dieter Söll

doi:10.1074/jbc.m411098200

Abstract

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 gamma-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 gamma-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.

Highlights

Two distinct pathways exist in nature for the formation of Gln-tRNAGln [1]
A threonine residue located in a flexible loop near the N terminus is used as the primary nucleophile by Mechanism of a tRNA-dependent Amidotransferase containing wild type (WT) and mutant GatD subunits in the presence of Glu-tRNAGln
When glutamate is activated by ␥-phosphorylation and ammonia is unavailable, the nucleophilic attack of ␥-phosphoryl-Glu-tRNAGln by the internal ␣-amino group (Scheme 1, step 4) prevails even under mild basic conditions, resulting in the formation of pyroglutamyltRNAGln

Summary

The abbreviations used are

GlnRS, glutaminyl-tRNA synthetase; GluRS, glutamyl-tRNA synthetase; AdT, tRNA-dependent amidotransplasm and a few bacteria, most extant organisms including all Archaea and the majority of bacteria lack a functional GlnRS and cannot attach glutamine directly to tRNAGln. Sequence analyses indicate that GatA is homologous to amidases and that GatD is similar to asparaginases [5, 6] Both amidases and asparaginases belong to the glutaminase superfamily that catalyzes amide bond hydrolysis and the subsequent transfer of ammonia to a wide variety of amide acceptors [7]. The validity of this intermediate was cast in doubt when recent attempts [10] failed to trap the intermeferase; GluAdT, Glu-tRNAGln-dependent AdT; HPLC, high pressure liquid chromatography; TLC, thin layer chromatography. Diate with purified GatCAB enzymes. Here we present our studies on the mechanism of tRNA-dependent transamidation using the archaeal-specific GluAdT GatDE as a model system

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION