Mechanism of a GatCAB Amidotransferase:  Aspartyl-tRNA Synthetase Increases Its Affinity for Asp-tRNA^Asn and Novel Aminoacyl-tRNA Analogues Are Competitive Inhibitors

Abstract

The trimeric GatCAB aminoacyl-tRNA amidotransferases catalyze the amidation of Asp-tRNAAsn and/or Glu-tRNAGln to Asn-tRNAAsn and/or Gln-tRNAGln, respectively, in bacteria and archaea lacking an asparaginyl-tRNA synthetase and/or a glutaminyl-tRNA synthetase. The two misacylated tRNA substrates of these amidotransferases are formed by the action of nondiscriminating aspartyl-tRNA synthetases and glutamyl-tRNA synthetases. We report here that the presence of a physiological concentration of a nondiscriminating aspartyl-tRNA synthetase in the transamidation assay decreases the Km of GatCAB for Asp-tRNAAsn. These conditions, which were practical for the testing of potential inhibitors of GatCAB, also allowed us to discover and characterize two novel inhibitors, aspartycin and glutamycin. These analogues of the 3'-ends of Asp-tRNA and Glu-tRNA, respectively, are competitive inhibitors of the transamidase activity of Helicobacter pylori GatCAB with respect to Asp-tRNAAsn, with Ki values of 134 microM and 105 microM, respectively. Although the 3' end of aspartycin is similar to the 3' end of Asp-tRNAAsn, this analogue was neither phosphorylated nor transamidated by GatCAB. These novel inhibitors could be used as lead compounds for designing new types of antibiotics targeting GatCABs, since the indirect pathway for Asn-tRNAAsn or Gln-tRNAGln synthesis catalyzed by these enzymes is not present in eukaryotes and is essential for the survival of the above-mentioned bacteria.

Methanothermobacter thermautotrophicus tRNAGln Confines the Amidotransferase GatCAB to Asparaginyl-tRNAAsn Formation

“…It is speculated that in vivo complexes between NDaaRSs and AdTs protect the aa-tRNA from deacylation. 22,32,33 The M. thermautotrophicus GatDE utilized both amide donors (Fig. 1a), though it is approximately fivefold more efficient with Asn than Gln as the donor for transamidating Glu-tRNA Gln ( Table 1).…”

Gln-tRNAGln synthesis in a dynamic transamidosome from Helicobacter pylori, where GluRS2 hydrolyzes excess Glu-tRNAGln

“…Despite this, in vivo experiments have shown that under certain conditions, the Glu residue of Glu-tRNA Gln can be incorporated into proteins, suggesting that an additional locking mechanism may be needed to prevent translational errors (6,14). Such a mechanism was shown in Pseudomonas aeruginosa and H. pylori , where the channeling of misacylated aa-tRNAs from the ND-aaRS to the aa-tRNA amidotransferase effectively sequesters them (15), as initially suggested by Schön et al (16). Sequestration of misacylated aa-tRNAs through the formation of more robust interactions was then confirmed in thermophilic organisms.…”

“…Cells for the overproduction of H. pylori GluRS2 and GatCAB were cultured and lysed as adapted from previous works (6,15). Both enzymes were purified by ion-exchange chromatography on DEAE-cellulose (DE52, Whatman), followed by affinity chromatography on Ni-NTA resin (Novagen).…”

“…For experiments measuring only transamidation, GatCAB was used at a concentration of 15 nM, while GluRS2 was used at 6.6 µM when it was in excess. The Glu-tRNA Gln substrate was synthesized separately, and purified as described (15). For reactions where glutamylation and transamidation were measured simultaneously, an equimolar concentration of GatCAB and GluRS2 (50 nM), and uncharged tRNA Gln at various concentrations were used.…”

“…Both kinds of experiment were performed at 37°C, with 10 µl reaction aliquots prepared and then analyzed by migration on cellulose thin-layer chromatography plates as previously described (24). Product formation was quantified using radioactivity standards (15). …”