Abstract

The amide aminoacyl-tRNAs, Gln-tRNAGln and Asn-tRNA Ans, are formed in many bacteria by a pretranslational tRNA-dependent amidation of the mischarged tRNA species, GlutRNAGln or Asp-tRNAAsn. This conversion is catalyzed by a heterotrimeric amidotransferase GatCAB in the presence of ATP and an amide donor (Gln or Asn). Helicobacter pylori has a single GatCAB enzyme required in vivo for both Gln-tRNAGln and Asn-tRNAAsn synthesis. In vitro characterization reveals that the enzyme transamidates Asp-tRNAAsn and Glu-tRNAGln with similar efficiency (kcat/K m of 1368.4 s-1/mM and 3059.3 s-1/mM respectively). The essential glutaminase activity of the enzyme is a property of the A-subunit, which displays the characteristic amidase signature sequence. Mutations of the GatA catalytic triad residues (Lys52, Ser 128, Ser152) abolished glutaminase activity and consequently the amidotransferase activity with glutamine as the amide donor. However, the latter activity was rescued when the mutant enzymes were presented with ammonium chloride. The presence of Asp-tRNAAsn and ATP enhances the glutaminase activity about 22-fold. H. pylori GatCAB uses the amide donor glutamine 129-fold more efficiently than asparagine, suggesting that GatCAB is a glutamine-dependent amidotransferase much like the unrelated asparagine synthetase B. Genomic analysis suggests that most bacteria synthesize asparagine in a glutamine-dependent manner, either by a tRNA-dependent or in a tRNA-independent route. However, all known bacteria that contain asparagine synthetase A form Asn-tRNAAsn by direct acylation catalyzed by asparaginyl-tRNA synthetase. Therefore, bacterial amide aminoacyl-tRNA formation is intimately tied to amide amino acid metabolism. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.