Transfer RNA-dependent amino acid biosynthesis: An essential route to asparagine formation

Abstract

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-tRNAs: setting the limits of the genetic code

“…Biochemical and genetic studies demonstrated that the tRNA-dependent Asp → Asn conversion is the only route to asparagine biosynthesis available in these organisms. Genome sequence analysis revealed that unless another still unknown enzyme for Asp → Asn conversion exists, Asp/ Glu-AdT is essential for asparagine synthesis in many organisms (Min et al 2002). These data lend support to the notion that the transamidation pathways are intimately linked to amino acid metabolism, as has also been proposed for other aspects of translation (Francklyn 2003; see also below).…”

Expanding tRNA recognition of a tRNA synthetase by a single amino acid change

“…However, an evolutionary change from D-AspRS to ND-AspRS may have also been logical, if one considers that Asp-tRNA Asn may not be as toxic to cellular protein synthesis as had been expected (B. Min, personal communication). The presence of a ND-AspRS may be beneficial in other ways, for instance, in case an organism needs to make asparagine (13). Therefore, it may be advantageous for an organism to retain ''flexibility'' with tRNA discrimination conversions in either direction, possibly in response to codon-usage drift or changing metabolic environments.…”

“…The archaeal genre comprises all archaeal AspRS enzymes, as well as one of two types of AspRSs found in the bacteria Thermus thermophilus (12), Deinococcus radiodurans (13), and Clostridium acetobutylicum. Whereas the archaeal AspRS enzymes were thought to be nondiscriminating (12,14), it is now known that in several archaea (including Thermococcus kodakaraensis), this enzyme is discriminating (15).…”

Distinguishing features of δ-proteobacterial genomes

“…(Planctomycetales). Among these species, it has been shown that the genome of D. radiodurans does not encode the regular enzymes for asparagine biosynthesis (asnA or asnB) and that Asp-tRNA Asn transamidation is the only pathway by which D. radiodurans can synthesize asparagine (28). The coexistence of GatCAB with AsnRS and GlnRS might then be explained in the nine organisms where GatCAB coexists with AsnRS and GlnRS by a role of GatBCA in the biosynthesis of asparagine or glutamine.…”