title: The nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori: anticodon-binding domain mutations that impact tRNA specificity and heterologous toxicity.
abstract: Divergent tRNA substrate recognition patterns distinguish the two distinct forms of aspartyl-tRNA synthetase (AspRS) that exist in different bacteria. In some cases, a canonical, discriminating AspRS (D-AspRS) specifically generates Asp-tRNA(Asp) and usually coexists with asparaginyl-tRNA synthetase (AsnRS). In other bacteria, particularly those that lack AsnRS, AspRS is nondiscriminating (ND-AspRS) and generates both Asp-tRNA(Asp) and the noncanonical, misacylated Asp-tRNA(Asn); this misacylated tRNA is subsequently repaired by the glutamine-dependent Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase (Asp/Glu-Adt). The molecular features that distinguish the closely related bacterial D-AspRS and ND-AspRS are not well-understood. Here, we report the first characterization of the ND-AspRS from the human pathogen Helicobacter pylori (H. pylori or Hp). This enzyme is toxic when heterologously overexpressed in Escherichia coli. This toxicity is rescued upon coexpression of the Hp Asp/Glu-Adt, indicating that Hp Asp/Glu-Adt can utilize E. coli Asp-tRNA(Asn) as a substrate. Finally, mutations in the anticodon-binding domain of Hp ND-AspRS reduce this enzyme's ability to misacylate tRNA(Asn), in a manner that correlates with the toxicity of the enzyme in E. coli.
authors.affiliation: Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
authors.forename: Pitak
authors.initials: P
authors.lastname: Chuawong
authors.forename: Tamara L
authors.initials: TL
authors.lastname: Hendrickson
mesh_terms: D000595:Amino Acid Sequence; D000926:Anticodon / Q000235:genetics*; D001226:Aspartate-tRNA Ligase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016480:Helicobacter pylori / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000633:toxicity; D012343:RNA, Transfer / Q000633:toxicity*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D000926:Anticodon; D001426:Bacterial Proteins; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D012343:RNA, Transfer; D001226:Aspartate-tRNA Ligase