Asp-to-Asn Substitution at the First Position of the DxD TOPRIM Motif of Recombinant Bacterial Topoisomerase I Is Extremely Lethal to E. coli

Bokun Cheng, Thirunavukkarasu Annamalai, Elena Sorokin, Maria Abrenica, Sandra Aedo, Yuk Ching Tse-Dinh

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

The TOPRIM domain found in many nucleotidyl transferases contains a DxD motif involved in magnesium ion coordination for catalysis. Medium- to high-copy-number plasmid clones of Yersinia pestis topoisomerase I (YpTOP) with Asp-to-Asn substitution at the first aspartate residue (D117N) of this motif could not be generated in Escherichia coli without second-site mutation even when expression was under the control of the tightly regulated BAD promoter and suppressed by 2% glucose in the medium. Arabinose induction of a single-copy YpTOP-D117N mutant gene integrated into the chromosome resulted in ∼ 105-fold of cell killing in 2.5 h. Attempt to induce expression of the corresponding E. coli topoisomerase I mutant (EcTOP-D111N) encoded on a high-copy-number plasmid resulted in either loss of viability or reversion of the clone to wild type. High-copy-number plasmid clones of YpTOP-D119N and EcTOP-D113N with the Asn substitution at the second Asp of the TOPRIM motif could be stably maintained, but overexpression also decreased cell viability significantly. The Asp-to-Asn substitutions at these TOPRIM residues can selectively decrease Mg2+ binding affinity with minimal disruption of the active-site geometry, leading to trapping of the covalent complex with cleaved DNA and causing bacterial cell death. The extreme sensitivity of the first TOPRIM position suggested that this might be a useful site for binding of small molecules that could act as topoisomerase poisons.

Original languageEnglish (US)
Pages (from-to)558-567
Number of pages10
JournalJournal of Molecular Biology
Volume385
Issue number2
DOIs
StatePublished - Jan 16 2009
Externally publishedYes

Keywords

  • DNA cleavage
  • DNA religation
  • TOPRIM
  • bactericidal
  • topoisomerase

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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