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PMID: 16800632 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

The nondiscriminating aspartyl-tRNA synthetase from Helicobacter pylori: anticodon-binding domain mutations that impact tRNA specificity and heterologous toxicity.

Biochemistry ·Vol. 45 ·No. 26 ·2006-07-04 ·Pages 8079-87

Chuawong P, Hendrickson TL

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.

MeSH Terms
Amino Acid Sequence Anticodon/genetics Aspartate-tRNA Ligase/chemistry,genetics,metabolism Bacterial Proteins/chemistry,genetics,metabolism Cloning, Molecular Conserved Sequence Escherichia coli/enzymology,genetics Helicobacter pylori/enzymology,genetics Kinetics Models, Molecular Molecular Sequence Data Protein Conformation RNA, Bacterial/toxicity RNA, Transfer/toxicity Recombinant Proteins/chemistry,metabolism Sequence Alignment Sequence Homology, Amino Acid
Chemicals
Anticodon Bacterial Proteins RNA, Bacterial Recombinant Proteins RNA, Transfer Aspartate-tRNA Ligase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Chuawong Pitak
Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Hendrickson Tamara L
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Article Info
Journal
Biochemistry
Abbr.
Biochemistry
ISSN
0006-2960
DOI
10.1021/bi060189c
Published
2006-07-04
Pages
8079-87
Language
English
Region
United States
NLM ID
0370623
PMCID
PMC2654173
Subset
IM
Grants
NIGMS NIH HHS · R01 GM071480 · United States
NIGMS NIH HHS · R01 GM071480-02 · United States
NIGMS NIH HHS · R01 GM071480-01A1 · United States
NIGMS NIH HHS · GM071480 · United States
NIGMS NIH HHS · R01 GM071480-03 · United States
External Links
PubMed source DOI: 10.1021/bi060189c PMC Full Text View journal details
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