期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：2
页码：382-387
DOI：10.1073/pnas.1423314112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe present structure reveals the architecture of the Pseudomonas aeruginosa bacterial-type asparagine-transamidosome, the most common macromolecular assembly required for asparaginyl-tRNAAsn formation in bacteria. We show that the presence of an additional GAD domain in the aspartyl-tRNA synthetase, common in most bacteria but missing in the archaeal-type Thermus thermophilus transamidosome, results in a complex with a distinct architecture and stoichiometry. Furthermore, our kinetic studies reveal that bacterial transamidosomes have distinct kinetic properties compared with the archaeal complex, with rapid release of the Asn-tRNAAsn product, leading to improved turnover by the bacterial-type aspartyl-tRNA synthetase in the complex. Overall, our study provides a structural basis for understanding tRNA-dependent asparagine biosynthesis found in the in majority of bacterial species. Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNAAsn, and instead synthesize asparagine from tRNAAsn-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNAAsn, and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNAAsn to form Asn-tRNAAsn for use in protein synthesis. ND-AspRS, GatCAB, and tRNAAsn may assemble in an [~]400-kDa complex, known as the Asn-transamidosome, which couples the two steps of asparagine biosynthesis in space and time to yield Asn-tRNAAsn. We report the 3.7-[IMG]f1.gif" ALT="A" BORDER="0"> resolution crystal structure of the Pseudomonas aeruginosa Asn-transamidosome, which represents the most common machinery for asparagine biosynthesis in bacteria. We show that, in contrast to a previously described archaeal-type transamidosome, a bacteria-specific GAD domain of ND-AspRS provokes a principally new architecture of the complex. Both tRNAAsn molecules in the transamidosome simultaneously serve as substrates and scaffolds for the complex assembly. This architecture rationalizes an elevated dynamic and a greater turnover of ND-AspRS within bacterial-type transamidosomes, and possibly may explain a different evolutionary pathway of GatCAB in organisms with bacterial-type vs. archaeal-type Asn-transamidosomes. Importantly, because the two-step pathway for Asn-tRNAAsn formation evolutionarily preceded the direct attachment of Asn to tRNAAsn, our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.
