Glutamyl-tRNA ^Gln amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis

Abstract

Asparaginyl-tRNA (Asn-tRNA) and glutaminyl-tRNA (Gln-tRNA) are essential components of protein synthesis. They can be formed by direct acylation by asparaginyl-tRNA synthetase (AsnRS) or glutaminyl-tRNA synthetase (GlnRS). The alternative route involves transamidation of incorrectly charged tRNA. Examination of the preliminary genomic sequence of the radiation-resistant bacterium Deinococcus radiodurans suggests the presence of both direct and indirect routes of Asn-tRNA and Gln-tRNA formation. Biochemical experiments demonstrate the presence of AsnRS and GlnRS, as well as glutamyl-tRNA synthetase (GluRS), a discriminating and a nondiscriminating aspartyl-tRNA synthetase (AspRS). Moreover, both Gln-tRNA and Asn-tRNA transamidation activities are present. Surprisingly, they are catalyzed by a single enzyme encoded by three ORFs orthologous to Bacillus subtilis gatCAB. However, the transamidation route to Gln-tRNA formation is idled by the inability of the discriminating D. radiodurans GluRS to produce the required mischarged Glu-tRNA Gln substrate. The presence of apparently redundant complete routes to Asn-tRNA formation, combined with the absence from the D. radiodurans genome of genes encoding tRNA-independent asparagine synthetase and the lack of this enzyme in D. radiodurans extracts, suggests that the gatCAB genes may be responsible for biosynthesis of asparagine in this asparagine prototroph.

Aminoacyl-tRNAs: setting the limits of the genetic code

“…The presence of the notionally redundant transamidation and direct acylation pathways in their active forms has also been observed, for example in Deinococcus radiodurans and Thermus thermophilus (Curnow et al 1998;Becker and Kern 1998). These organisms contain all possible enzymes for Asn-tRNA and Gln-tRNA formation, AsnRS, GlnRS and Asp/Glu-AdT.…”

The Nondiscriminating Aspartyl-tRNA Synthetase from Helicobacter pylori: Anticodon-Binding Domain Mutations That Impact tRNA Specificity and Heterologous Toxicity

“…Aspartyl-tRNA synthetase (AspRS) is an example of an AARS that can aminoacylate two different tRNA isoacceptors: tRNA Asp and tRNA Asn (5)(6)(7)12,3,14). In eukaryotes, some bacteria and some archaea, AspRS is a canonical, discriminating enzyme (D-AspRS) that only generates Asp-tRNA Asp (25,14).…”

“…In eukaryotes, some bacteria and some archaea, AspRS is a canonical, discriminating enzyme (D-AspRS) that only generates Asp-tRNA Asp (25,14). In other bacteria, archaea, and some organelles however, AspRS is a non-canonical, non-discriminating enzyme (ND-AspRS) that generates both AsptRNA Asp and the misacylated Asp-tRNA Asn (5)(6)(7)12,3,14). This Asp-tRNA Asn is converted to Asn-tRNA Asn by a glutamine-dependent Asp-tRNA Asn /Glu-tRNA Gln amidotransferase (Asp/ Glu-Adt) (26,27,9,28,12).…”

Evolutionary Divergence of the Archaeal Aspartyl-tRNA Synthetases into Discriminating and Nondiscriminating Forms

“…Concentrations of the AspRSs were 25 nM for M. thermautotrophicus AspRS, 10 nM for P. kodakaraensis AspRS, and 5 nM for F. acidarmanus AspRS. After incubation at the relevant temperature for 40 min, the substrate was isolated by phenol extraction and ethanol precipitation as described (15). The tRNA amidotransferase assay has been described also (15).…”

“…Preparation of Unfractionated tRNA-Unfractionated tRNA was purified from 5 g of M. marburgensis and P. furiosus cells as described (15) (18) was also cloned into the NdeI-SacI sites of pET20b without the stop codon of the gatB gene. All proteins were expressed from the E. coli BL21-CodonPlus(DE3)-RIL strain (Stratagene), which expresses the E. coli minor tRNA genes argU, ileY, and leuW from a chloramphenicol-resistant vector.…”