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.

Biochemical experiments and genomic sequence analysis showed that Deinococcus radiodurans and Thermus thermophilus do not possess asparagine synthetase (encoded by asnA or asnB), the enzyme forming asparagine from aspartate. Instead these organisms derive asparagine from asparaginyl-tRNA, which is made from aspartate in the tRNA-dependent transamidation pathway [ A sparagine, one of the 21 cotranslationally inserted amino acids that make up proteins, is known to be synthesized from aspartate in an ATP-dependent amidation reaction (1). Two mechanistically distinct asparagine synthetases are known (2-4). The one encoded by asnA utilizes ammonia as amide donor, whereas the asnB-derived protein works with glutamine. These enzymes are present in organisms of all domains, the major one being asparagine synthetase B, which is encoded in different organisms by a small number of related genes. Both enzymes are well studied biochemically (5), and their crystal structures are known (6, 7). Until recently they were assumed to be the sole biosynthetic route to asparagine. However, Thermus thermophilus and Deinococcus radiodurans lack these enzymes; instead they employ a tRNA-dependent transamidation mechanism for conversion of aspartate to asparagine (8,9).Two routes of Asn-tRNA synthesis exist in D. radiodurans (Fig. 1). Similar to many bacteria, Deinococcus contains a tRNA-dependent two-step pathway of Asn-tRNA formation. In the first step a nondiscriminating aspartyl-tRNA synthetase (AspRS)2 generates the misacylated Asp-tRNA Asn species, which then is amidated to the correctly charged Asn-tRNA Asn by the heterotrimeric Asp-tRNA Asn amidotransferase (Asp-AdT; encoded by the gatCAB genes) with glutamine serving as the amide donor (9). In addition, the organism also contains asparaginyl-tRNA synthetase (AsnRS; ref. 10), which is active and produces Asn-tRNA in the canonical aminoacylation reaction (9). The close Deinococcus relative T. thermophilus has similar enzymes and presumably uses the same asparagine biosynthetic routes (8,11,12). It was suggested earlier (8, 9) that the role of Asp-AdT in D. radiodurans and T. thermophilus is to synthesize the cell's entire supply of asparagine, because no asnA or asnB orthologs are present in the genome (10), and because biochemical analysis of crude cell extracts did not reveal the presence of any tRNA-independent asparagine synthetase activity (8, 9). Here we present data from D. radiodurans that prove this role to be correct and propose that tRNA-dependent asparagine synthesis occurs in many bacteria as the sole synthetic route to this essential amino acid. ͞10 g/ml methionine͞25 g/ml histidine͞30 g/ml cysteine͞1 g/ml nicotinic acid͞2 mg/ml fructose. Where necessary, the medium was supplemented with 10 g/ml kanamycin͞2.5 g/ml tetracycline͞3 g/ml chloramphenicol͞20 g/ml asparagine. E. coli strain DH5␣ was grown at 37°C on LB medium (1% tryptone͞0.5% yeast extract͞0.5% NaCl͞1.5% agar) supplemented where necessary with 50 g͞ml ampicillin and 30 g͞ml tetracycline. E. coli strain JF4...

Aminoacyl-tRNA is generally formed by aminoacyltRNA synthetases, a family of 20 enzymes essential for accurate protein synthesis. However, most bacteria generate one of the two amide aminoacyl-tRNAs, Asn-tRNA or Gln-tRNA, by transamidation of mischarged AsptRNA Asn or Glu-tRNA Gln catalyzed by a heterotrimeric amidotransferase (encoded by the gatA, gatB, and gatC genes). The Chlamydia trachomatis genome sequence reveals genes for 18 synthetases, whereas those for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase are absent. Yet the genome harbors three gat genes in an operon-like arrangement (gatCAB). We reasoned that Chlamydia uses the gatCAB-encoded amidotransferase to generate both Asn-tRNA and Gln-tRNA. C. trachomatis aspartyl-tRNA synthetase and glutamyltRNA synthetase were shown to be non-discriminating synthetases that form the misacylated tRNA Asn and tRNA Gln species. A preparation of pure heterotrimeric recombinant C. trachomatis amidotransferase converted Asp-tRNA Asn and Glu-tRNA Gln into Asn-tRNA and Gln-tRNA, respectively. The enzyme used glutamine, asparagine, or ammonia as amide donors in the presence of either ATP or GTP. These results suggest that C. trachomatis employs the dual specificity gatCAB-encoded amidotransferase and 18 aminoacyl-tRNA synthetases to create the complete set of 20 aminoacyl-tRNAs.

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