Characterization of the indirect pathway for Asn-tRNA(Asn) biosynthesis used by the human pathogen Helicobacter pylori

The aminoacylation of tRNAs is a crucial step in maintaining fidelity in protein biosynthesis. This task can be accomplished by a group of enzymes called aminoacyl-tRNA synthetases (AARSs). In some organisms, there are 20 AARSs, with one enzyme for each amino acid and tRNA(s) pair. However, several indirect aminoacylation pathways, that replace one or more AARS, have been characterized in archaea, some bacteria, and organelles. The most common indirect aminoacylation pathway is the synthesis of Gln-tRNAGln and Asn-tRNAAsn via transamidation of two misacylated tRNAs, Glu-tRNA Gln and Asp-tRNAAsn, respectively.; Helicobacter pylori (H. pylori) is a human pathogenic bacterium whose genome lacks genes encoding for asparaginyl-tRNA synthetase (AsnRS) and glutaminyl-tRNA synthetase (GlnRS); this bacterium relies on Asp-tRNA Asn/Glu-tRNAGln amidotransferase (Asp/Glu-Adt) activity in order to synthesize Asn-tRNAAsn and Gln-tRNAGln from mischarged Asp-tRNAAsn and Glu-tRNAGln. Furthermore, in order to generate these two misacylated aminoacyl-tRNAs as substrates for Asp/Glu-Adt, the tRNA specificities of two H. pylori AARSs are modified: This organism's non-discriminating aspartyl-tRNA synthetase (ND-AspRS) generates Asp-tRNAAsp and Asp-tRNA Asn, and H. pylori glutamyl-tRNA synthetase 2 (GluRS2) generates Glu-tRNAGln, specifically.; Here we report the first characterization of the ND-AspRS from H. pylori. This enzyme aminoacylates both tRNAAsp and tRNAAsn, however the cognate tRNAAsp is a better substrate. Overexpression of this ND-AspRS in E. coli results in a toxic phenotype, which can be rescued by co-expression of H. pylori Asp/Glu-Adt. Kinetic analyses of wild-type H. pylori AspRS as well as two mutations in the anticodon binding domain demonstrate that this toxicity is correlated to the specificity ratio of tRNAAsp /tRNAAsn. The determination of the mechanism behind the toxicity of this enzyme utilizing an E. coli trpA34, a tryptophan auxotroph suggests that this ND-AspRS is generating Asp-tRNAAsn in vivo and aspartate is being erroneously introduced into proteins at asparagine codons.; The heterotrimeric Asp/Glu-Adt from H. pylori was cloned and purified to homogeneity. This enzyme was shown to be catalytically active by using a newly developed thin layer electrophoresis (TLE) technique.; Finally, Hp0100, a protein with unknown function, has been successfully cloned, overexpressed, and purified to homogeneity. This protein enhances the deacylation of both Asp-tRNAAsp and Asp-tRNAAsn in vitro. This enhancement is even more pronounced in the presence of H. pylori ND-AspRS, an enzyme that is not known to have deacylation activity.