当前位置： SCI文献检索 > JOURNAL OF MOLECULAR BIOLOGY期刊下所有文献 > Asp-to-Asn substitution at the first position of the DxD TOPRIM motif of recombinant bacterial topoisomerase I is extremely lethal to E. coli.

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.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Cheng B,Annamalai T,Sorokin E,Abrenica M,Aedo S,Tse-Dinh YC

doi

10.1016/j.jmb.2008.10.073

subject

Has Abstract

pub_date

2009-01-16 00:00:00

pages

558-67

issue

2

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(08)01393-4

journal_volume

385

pub_type

杂志文章

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