Evolutionary divergence of the archaeal aspartyl-tRNA synthetases into discriminating and nondiscriminating forms

Asparaginyl-tRNA (Asn-tRNA) is generated in nature via two alternate routes, either direct acylation of tRNA with asparagine by asparaginyl-tRNA synthetase (AsnRS) or in a two-step pathway that requires misacylated Asp-tRNA^Asn as an intermediate. This misacylated aminoacyl-tRNA is formed by a nondiscriminating aspartyl-tRNA synthetase (AspRS), an enzyme that in addition to forming Asp-tRNA^Asp also misacylates tRNA^Asn. In contrast, a discriminating AspRS cannot acylate tRNA^Asn. It has been suggested that the archaeal AspRS enzymes are nondiscriminating, whereas the bacterial ones discriminate. The archaeal and bacterial AspRS proteins are indeed distinct in sequence and structure. However, we show that both discriminating and nondiscriminating forms of AspRS exist among the archaea. Using unfractionated methanobacterial and pyrococcal tRNA, the Methanothermobacter thermautotrophicus AspRS acylated approximately twice as much tRNA as did AspRS from Pyrococcus kodakaraensis or Ferroplasma acidarmanus. Proof that Asp-tRNA^Asn was generated by the methanogen synthetase was the conversion of Asp-tRNA formed by M. thermautotrophicus AspRS to Asn-tRNA by M. thermautotrophicus Asp-tRNA^Asn amidotransferase. In contrast, Asp-tRNA formed by the Pyrococcus or Ferroplasma enzymes was not a substrate for the amidotransferase. Also, although all three AspRS enzymes charged tRNA^Asp transcripts, only M. thermautotrophicus AspRS aspartylated the tRNA^Asn transcript. Genomic analysis provides a rationale for the nature of these enzymes. The mischarging AspRS correlates with the absence in the genome of AsnRS and the presence of Asp-tRNA^Asn amidotransferase, employed by the transamidation pathway. In contrast, the discriminating AspRS correlates with the absence of the amidotransferase and the presence of AsnRS, forming Asn-tRNA by direct aminoacylation. The high sequence identity, up to 60% between discriminating and nondiscriminating archaeal AspRSs, suggests that few mutational steps may be necessary to convert the tRNA-discriminating ability of a tRNA synthetase.