The heterotrimeric Thermus thermophilus Asp-tRNA(Asn) amidotransferase canalso generate Gln-tRNA(Gln)

Tlermas thermophilus strain HB8 is known to have a heterodimeric aspartyl-t RNA(Asn) amidotransferase (Asp-AdT) capable of forming Asn-tRNA(Asn) [Becke r, H.D. and Kern, D. (1998) Proc. Natl, Acad. Sci. USA 95, 12832-12837]. He re we show that, like other bacteria, T. thermophilus possesses the canonic al set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpres sion in Escherichia coli of the thermophilic holoenzyme. The overproduced T . thermophilus AdT can generate Gln-tRNA(Gln) as well as Asn-tRNA(Asn). Thu s, the T. thermophilus tRNA-dependent AdT is a dual-specific Asp/ Glu-AdT r esembling other bacterial AdTs. In addition, rye observed that removal of t he 44 carboxy-terminal amino acids of the GatA subunit only inhibits the As p-AdT activity, leaving the Glu-AdT activity of the mutant AdT unaltered: t his shows that Asp-AdT and Glu-AdT activities can be mechanistically separa ted. (C) 2000 Federation of European Biochemical Societies, Published by El sevier Science B.V. All rights reserved.