Suzuki Tateki, Nakamura Akiyoshi, Kato Koji, Söll Dieter, Tanaka Isao, Sheppard Kelly, Yao Min
Graduate School of Life Science and.
Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan; Department of Molecular Biophysics and Biochemistry and Department of Chemistry, Yale University, New Haven, CT 06520; and.
Proc Natl Acad Sci U S A. 2015 Jan 13;112(2):382-7. doi: 10.1073/pnas.1423314112. Epub 2014 Dec 29.
Many prokaryotes lack a tRNA synthetase to attach asparagine to its cognate tRNA(Asn), and instead synthesize asparagine from tRNA(Asn)-bound aspartate. This conversion involves two enzymes: a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) that forms Asp-tRNA(Asn), and a heterotrimeric amidotransferase GatCAB that amidates Asp-tRNA(Asn) to form Asn-tRNA(Asn) for use in protein synthesis. ND-AspRS, GatCAB, and tRNA(Asn) 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-tRNA(Asn). We report the 3.7-Å 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 tRNA(Asn) 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-tRNA(Asn) formation evolutionarily preceded the direct attachment of Asn to tRNA(Asn), our structure also may reflect the mechanism by which asparagine was initially added to the genetic code.
许多原核生物缺乏将天冬酰胺连接到其同源tRNA（Asn）上的tRNA合成酶，而是从与tRNA（Asn）结合的天冬氨酸合成天冬酰胺。这种转化涉及两种酶：一种非特异性天冬氨酰-tRNA合成酶（ND-AspRS），它形成天冬氨酰-tRNA（Asn）；以及一种异源三聚体酰胺转移酶GatCAB，它将天冬氨酰-tRNA（Asn）酰胺化形成用于蛋白质合成的天冬氨酰-tRNA（Asn）。ND-AspRS、GatCAB和tRNA（Asn）可能组装成一个约400 kDa的复合物，称为天冬酰胺转氨体，它在空间和时间上耦合天冬酰胺生物合成的两个步骤，以产生天冬氨酰-tRNA（Asn）。我们报告了铜绿假单胞菌天冬酰胺转氨体的3.7 Å分辨率晶体结构，它代表了细菌中天冬酰胺生物合成最常见的机制。我们表明，与先前描述的古细菌型转氨体相反，ND-AspRS的细菌特异性GAD结构域引发了复合物主要的新结构。转氨体中的两个tRNA（Asn）分子同时作为复合物组装的底物和支架。这种结构合理化了细菌型转氨体中ND-AspRS更高的动态性和更大的周转率，并且可能解释了在具有细菌型与古细菌型天冬酰胺转氨体的生物体中GatCAB不同的进化途径。重要的是，由于天冬氨酰-tRNA（Asn）形成的两步途径在进化上先于天冬酰胺直接连接到tRNA（Asn），我们的结构也可能反映了天冬酰胺最初被添加到遗传密码中的机制。
