title: Structure of the Pseudomonas aeruginosa transamidosome reveals unique aspects of bacterial tRNA-dependent asparagine biosynthesis.
abstract: 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.
authors.affiliation: Graduate School of Life Science and.
authors.forename: Tateki
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: 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.
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Nakamura
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan;
authors.forename: Koji
authors.initials: K
authors.lastname: Kato
authors.affiliation: Department of Molecular Biophysics and Biochemistry and Department of Chemistry, Yale University, New Haven, CT 06520; and dieter.soll@yale.edu yao@castor.sci.hokudai.ac.jp.
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan;
authors.forename: Isao
authors.initials: I
authors.lastname: Tanaka
authors.affiliation: Department of Chemistry, Skidmore College, Saratoga Springs, NY 12866.
authors.forename: Kelly
authors.initials: K
authors.lastname: Sheppard
authors.affiliation: Graduate School of Life Science and Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan; dieter.soll@yale.edu yao@castor.sci.hokudai.ac.jp.
authors.forename: Min
authors.initials: M
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000096:biosynthesis*; D001226:Aspartate-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D012348:RNA, Transfer, Asn / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism; D046249:Transfer RNA Aminoacylation / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D012348:RNA, Transfer, Asn; D001216:Asparagine; D001226:Aspartate-tRNA Ligase
keywords: GatCAB; asparagine biosynthesis; aspartyl-tRNA synthetase; transamidosome