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 Citations (Scopus)

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

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Type I DNA Topoisomerase
Yersinia pestis
Escherichia coli
Plasmids
Clone Cells
Bacterial DNA
Arabinose
Poisons
Viperidae
Transferases
Catalysis
Aspartic Acid
Magnesium
Catalytic Domain
Cell Survival
Cell Death
Chromosomes
Binding Sites
Ions
Glucose

Keywords

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

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

Asp-to-Asn Substitution at the First Position of the DxD TOPRIM Motif of Recombinant Bacterial Topoisomerase I Is Extremely Lethal to E. coli. / Cheng, Bokun; Annamalai, Thirunavukkarasu; Sorokin, Elena; Abrenica, Maria; Aedo, Sandra; Tse-Dinh, Yuk Ching.

In: Journal of Molecular Biology, Vol. 385, No. 2, 16.01.2009, p. 558-567.

Research output: Contribution to journalArticle

Cheng, Bokun ; Annamalai, Thirunavukkarasu ; Sorokin, Elena ; Abrenica, Maria ; Aedo, Sandra ; Tse-Dinh, Yuk Ching. / Asp-to-Asn Substitution at the First Position of the DxD TOPRIM Motif of Recombinant Bacterial Topoisomerase I Is Extremely Lethal to E. coli. In: Journal of Molecular Biology. 2009 ; Vol. 385, No. 2. pp. 558-567.
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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.",
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