title: Asp-to-Asn substitution at the first position of the DxD TOPRIM motif of recombinant bacterial topoisomerase I is extremely lethal to E. coli.
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 approximately 10(5)-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 Mg(2+) 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Basic Science Building, New York Medical College, Valhalla, NY 10595, USA.
authors.forename: Bokun
authors.initials: B
authors.lastname: Cheng
authors.forename: Thirunavukkarasu
authors.initials: T
authors.lastname: Annamalai
authors.forename: Elena
authors.initials: E
authors.lastname: Sorokin
authors.forename: Maria
authors.initials: M
authors.lastname: Abrenica
authors.forename: Sandra
authors.initials: S
authors.lastname: Aedo
authors.forename: Yuk-Ching
authors.initials: YC
authors.lastname: Tse-Dinh
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D050296:Microbial Viability; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D015010:Yersinia pestis / Q000201:enzymology*
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D004264:DNA Topoisomerases, Type I