Enzymological characterization of the nuclease domain from the bacterial toxin colicin E9 from Escherichia coli

The cytotoxicity of the bacterial toxin colicin E9 is due to a non-specific DNase that penetrates the cytoplasm of the infected organism and causes cell death. We report the first enzymological characterization of the overexpressed and purified 15 kDa DNase domain (E9 DNase) from this class of toxin...

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Bibliographic Details
Published in:Biochemical journal 1998-09, Vol.334 ( Pt 2) (2), p.387-392
Main Authors: Pommer, A J, Wallis, R, Moore, G R, James, R, Kleanthous, C
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
Circular Dichroism
Colicins - chemistry
Colicins - metabolism
Deoxyribonuclease I - metabolism
Deoxyribonucleases - chemistry
Deoxyribonucleases - metabolism
DNA - metabolism
Escherichia coli - enzymology
Escherichia coli Proteins
Kinetics
Magnesium - pharmacology
Plasmids
Protein Conformation
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Summary:The cytotoxicity of the bacterial toxin colicin E9 is due to a non-specific DNase that penetrates the cytoplasm of the infected organism and causes cell death. We report the first enzymological characterization of the overexpressed and purified 15 kDa DNase domain (E9 DNase) from this class of toxin. CD spectroscopy shows the E9 DNase to be structured in solution, and analytical ultracentrifugation data indicate that the enzyme is a monomer. The nuclease activity of the E9 DNase was compared with the well-studied, non-specific DNase I by using a spectrophotometric assay with calf thymus DNA as the substrate. Both enzymes require divalent metal ions for activity but, unlike DNase I, the E9 DNase is not activated by Ca2+ ions. Somewhat surprisingly, the E9 DNase shows optimal activity and linear kinetics in the presence of transition metals such as Ni2+ and Co2+ but displays non-linear kinetics with metals such as Mg2+ and Ca2+. Conversely, Ni2+ and other transition metals showed poor activity in a plasmid-based nicking assay, yielding significant amounts of linearized plasmid, whereas Mg2+ was very active, with the main intermediate being open-circle DNA. The results suggest that, on entry into bacterial cells, the E9 DNase is likely to exhibit primarily Mg2+-dependent nicking activity against chromosomal DNA, although other metals could also be utilized to introduce both single- and double-strand cleavages.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj3340387