Abstract
The amide aminoacyl-tRNAs, Gln-tRNA(Gln) and Asn-tRNA(Asn), are formed in many bacteria by a pretranslational tRNA-dependent amidation of the mischarged tRNA species, Glu-tRNA(Gln) or Asp-tRNA(Asn). 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-tRNA(Gln) and Asn-tRNA(Asn) synthesis. In vitro characterization reveals that the enzyme transamidates Asp-tRNA(Asn) and Glu-tRNA(Gln) with similar efficiency (k(cat)/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 (Lys(52), Ser(128), Ser(152)) 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-tRNA(Asn) 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-tRNA(Asn) by direct acylation catalyzed by asparaginyl-tRNA synthetase. Therefore, bacterial amide aminoacyl-tRNA formation is intimately tied to amide amino acid metabolism.
Highlights
The accurate pairing of an amino acid with its cognate tRNA is essential for the fidelity of protein biosynthesis
In prokaryotes lacking a glutaminyl-tRNA synthetase (GlnRS), a non-discriminating glutamyl-tRNA synthetase (NDGluRS) [3] forms the misacylated Glu-tRNAGln, which is subsequently converted by amidation to Gln-tRNAGln catalyzed by a glutamyl-tRNAGln amidotransferase (Glu-AdT) in the presence of an amide donor (4 – 6)
Asp-tRNAAsn and Glu-tRNAGln Are Good Substrates for H. pylori GatCAB—Our results demonstrate that H. pylori GatCAB is about as efficient as an aspartyl-tRNAAsn amidotransferase (Asp-AdT) as it is a Glu-AdT; this is plausible when one considers that in vivo this enzyme is expected to transamidate both Glu-tRNAGln and AsptRNAAsn
Summary
General—All oligonucleotide synthesis and DNA sequencing was carried out by the Keck Foundation Biotechnology Research Laboratory at Yale University. [L-14C]glutamine (224 mCi/mmol) was from American Radiolabeled Chemicals The reaction conditions were as follows: 50 mM Hepes-NaOH, pH 7.2, with 25 mM KCl, 15 mM MgCl2, 4 mM ATP, 5 mM DTT, 0.22 mg/ml P. aeruginosa AspRS, 1 mM L-Asp, 21 M unlabeled H. pylori tRNAAsn, and 1.33 M 32P-labeled H. pylori tRNAAsn. The samples were phenol/chloroform-extracted, and excess ATP was removed as described previously [11] Samples with only cold tRNAAsn were done in parallel to be used in the glutaminase assays (see below). Glutaminase Assays—The kinetic parameters of the glutaminase activity of the H. pylori GatCAB were carried out at 37 °C in 1ϫ AdT buffer in the presence or absence of 4 mM ATP and or 10 –11 M unlabeled mischarged tRNA (Asp-tRNAAsn or Glu-tRNAGln). Care was taken to ensure that all members in a genus had the same content of the relevant genes
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