In prokaryotes, mono ADP-ribose transfer enzymes represent a family of exotoxins that display activity in a variety of bacterial pathogens responsible for causing disease in plants and animals, including those affecting mankind such as diphtheria, cholera and whooping cough. We report here that NarE, a putative ADP-ribosylating toxin previously identified from Neisseria meningitidis, which shares structural homologies with Escherichia coli heat labile enterotoxin and toxin from Vibrio cholerae, possesses an iron-sulfur centre. The recombinant protein was expressed in E. coli and when purified at high concentration NarE has a distinctive golden brown in color. Evidence from UV-visible spectrophotometry and EPR spectroscopy revealed characteristics consistent of an iron-binding protein. The presence of iron was determined by colorimetric method and by Atomic Absorption Spectrophotometer. To obtain information about the identity of the amino-acids involved in binding iron, a combination of site-directed mutagenesis, UV-vis and enzymatic assays were performed. All four cysteine residues were individually replaced by serine. Substitution of C67 and C128 into serine caused a drastic reduction in the UV-vis signal intensity and in the A420/A280 ratio, suggesting that these two residues interact directly with the cluster and are essential for the formation of a stable coordination. This modification led to a consistent loss in ADP-ribosyltransferase activity while decrease in NAD-glycohydrolase was less dramatic in the C67S and C128S mutants indicating that the correct assembly of the iron-binding site is essential for exploiting transferase but not hydrolase activity. This is the first observation that a member of the ADP-ribosyltransferase family is an iron dependent enzyme and that contains a Fe-S cluster implicated in catalysis. This novel discovery may lead to the discovery of novel functions exerted by this class of enzymes.
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|Titolo:||Identification of an Iron-sulfur Cluster that modulates the Enzymatic Activity in NarE, a Neisseria Meningitidis ADP-ribosyltransferase|
|Citazione:||M., D.V., Pogni, R., Baratto, M.C., A., N., R., R., M., P., et al. (2009). Identification of an Iron-sulfur Cluster that modulates the Enzymatic Activity in NarE, a Neisseria Meningitidis ADP-ribosyltransferase. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 284(48), 33040-33047.|
|Appare nelle tipologie:||1.1 Articolo in rivista|