Becker H D, Min B, Jacobi C, Raczniak G, Pelaschier J, Roy H, Klein S, Kern D, Söll D
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
FEBS Lett. 2000 Jul 7;476(3):140-4. doi: 10.1016/s0014-5793(00)01697-5.
Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl-tRNA(Asn) amidotransferase (Asp-AdT) capable of forming Asn-tRNA(Asn) [Becker, H.D. and Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832-12837]. Here we show that, like other bacteria, T. thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme. The overproduced T. thermophilus AdT can generate Gln-tRNA(Gln) as well as Asn-tRNA(Asn). Thus, the T. thermophilus tRNA-dependent AdT is a dual-specific Asp/Glu-AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy-terminal amino acids of the GatA subunit only inhibits the Asp-AdT activity, leaving the Glu-AdT activity of the mutant AdT unaltered; this shows that Asp-AdT and Glu-AdT activities can be mechanistically separated.
嗜热栖热菌HB8菌株已知具有一种异源二聚体天冬氨酰 - tRNA（Asn）酰胺转移酶（Asp - AdT），能够形成Asn - tRNA（Asn）[贝克尔，H.D.和克恩，D.（1998年）《美国国家科学院院刊》95，12832 - 12837]。在此我们表明，与其他细菌一样，嗜热栖热菌拥有一套典型的酰胺转移酶（AdT）基因（gatA、gatB和gatC）。我们克隆并测序了这些基因，并构建了一个人工操纵子，用于在大肠杆菌中过表达嗜热全酶。过量产生的嗜热栖热菌AdT能够生成Gln - tRNA（Gln）以及Asn - tRNA（Asn）。因此，嗜热栖热菌依赖tRNA的AdT是一种双特异性的Asp/Glu - AdT，类似于其他细菌的AdT。此外，我们观察到去除GatA亚基的44个羧基末端氨基酸仅抑制Asp - AdT活性，而突变型AdT的Glu - AdT活性未改变；这表明Asp - AdT和Glu - AdT活性在机制上可以分开。
