The Helicobacter pylori amidotransferase GatCAB is equally efficient in glutamine-dependent transamidation of Asp-tRNAAsn and Glu-tRNA Gln

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