On the evolution of the tRNA-dependent amidotransferases, GatCAB and GatDE

Glutaminyl-tRNA synthetase and asparaginyl-tRNA synthetase evolved from glutamyl-tRNA synthetase and aspartyl-tRNA synthetase, respectively, after the split in the last universal communal ancestor (LUCA). Glutaminyl-tRNA^Gln and asparaginyl-tRNA^Asn were likely formed in LUCA by amidation of the mischarged species, glutamyl-tRNA^Gln and aspartyl-tRNA^Asn, by tRNA-dependent amidotransferases, as is still the case in most bacteria and all known archaea. The amidotransferase GatCAB is found in both domains of life, while the heterodimeric amidotransferase GatDE is found only in Archaea. The GatB and GatE subunits belong to a unique protein family that includes Pet112 that is encoded in the nuclear genomes of numerous eukaryotes. GatE was thought to have evolved from GatB after the emergence of the modern lines of decent. Our phylogenetic analysis though places the split between GatE and GatB, prior to the phylogenetic divide between Bacteria and Archaea, and Pet112 to be of mitochondrial origin. In addition, GatD appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, while GatDE is an archaeal signature protein, it likely was present in LUCA together with GatCAB. Archaea retained both amidotransferases, while Bacteria emerged with only GatCAB. The presence of GatDE has favored a unique archaeal tRNA^Gln that may be preventing the acquisition of glutaminyl-tRNA synthetase in Archaea. Archaeal GatCAB, on the other hand, has not favored a distinct tRNA^Asn, suggesting that tRNA^Asn recognition is not a major barrier to the retention of asparaginyl-tRNA synthetase in many Archaea.     

Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

Many bacteria form Gln-tRNA^Gln and Asn-tRNA^Asn by conversion of the misacylated Glu-tRNA^Gln and Asp-tRNA^Asn species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction.A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn²⁺ site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn²⁺ binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA^Gln or Asn-tRNA^Asn.     

The role of the catalytic domain of E. coli GluRS in tRNA discrimination

Discrimination of tRNA^Gln is an integral function of several bacterial glutamyl-tRNA synthetases (GluRS). The origin of the discrimination is thought to arise from unfavorable interactions between tRNA^Gln and the anticodon-binding domain of GluRS. From experiments on an anticodon-binding domain truncated Escherichia coli (E. coli) GluRS (catalytic domain) and a chimeric protein, constructed from the catalytic domain of E. coli GluRS and the anticodon-binding domain of E. coli glutaminyl-tRNA synthetase (GlnRS), we show that both proteins discriminate against E. coli tRNA^Gln. Our results demonstrate that in addition to the anticodon-binding domain, tRNA^Gln discriminatory elements may be present in the catalytic domain in E. coli GluRS as well.     

Redox status affects the catalytic activity of glutamyl-tRNA synthetase

Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles in tetrapyrrole biosynthesis is not known. Previous studies have shown that GluRS1, one of two GluRSs from the extremophile Acidithiobacillus ferrooxidans, is inactivated when intracellular heme is elevated suggesting a specific role for GluRS1 in the regulation of tetrapyrrole biosynthesis. We now show that, in vitro, GluRS1 activity is reversibly inactivated upon oxidation by hemin and hydrogen peroxide. The targets for oxidation-based inhibition were found to be cysteines from a SWIM zinc-binding motif located in the tRNA acceptor helix-binding domain. tRNA^Glu was able to protect GluRS1 against oxidative inactivation by hemin plus hydrogen peroxide. The sensitivity to oxidation of A. ferrooxidans GluRS1 might provide a means to regulate tetrapyrrole and protein biosynthesis in response to extreme changes in both the redox and heme status of the cell via a single enzyme.