Many prokaryotes form the amide aminoacyl-tRNAs glutaminyl-tRNA and asparaginyl-tRNA by tRNA-dependent amidation of the mischarged tRNA species, glutamyl-tRNA(Gln) or aspartyl-tRNA(Asn). Archaea employ two such amidotransferases, GatCAB and GatDE, while bacteria possess only one, GatCAB. The Methanothermobacter thermautotrophicus GatDE is slightly more efficient using Asn as an amide donor than Gln (k(cat)/K(M) of 5.4 s(-1)/mM and 1.2 s(-1)/mM, respectively). Unlike the bacterial GatCAB enzymes studied to date, the M. thermautotrophicus GatCAB uses Asn almost as well as Gln as an amide donor (k(cat)/K(M) of 5.7 s(-1)/mM and 16.7 s(-1)/mM, respectively). In contrast to the initial characterization of the M. thermautotrophicus GatCAB as being able to form Asn-tRNA(Asn) and Gln-tRNA(Gln), our data demonstrate that while the enzyme is able to transamidate Asp-tRNA(Asn) (k(cat)/K(M) of 125 s(-1)/mM) it is unable to transamidate M. thermautotrophicus Glu-tRNA(Gln). However, M. thermautotrophicus GatCAB is capable of transamidating Glu-tRNA(Gln) from H. pylori or B. subtilis, and M. thermautotrophicus Glu-tRNA(Asn). Thus, M. thermautotrophicus encodes two amidotransferases, each with its own activity, GatDE for Gln-tRNA and GatCAB for Asn-tRNA synthesis.