NARE: AN IRON-SULFUR CLUSTER-DEPENDENT MONO ADP-RIBOSYLTRANSFERASE FROM NEISSERIA MENINGITIDIS

Mono ADP-ribosylation is a covalent and enzyme-catalyzed reaction in which β-nicotinamide adenine dinucleotide (NAD+), acts as substrate. In prokaryotes, mono ADP-ribose transfer enzymes, like their eukaryotic counterpart catalyze a selective transfer of the ADP-ribose moiety to specific amino-acids in protein targets with release of nicotinamide. A computer-based-strategy allowed us to identify NarE (Neisseria ADP-ribosylating enzyme), a previously unidentified ADP-ribosyltransferase, in strain MC58 of the gram-negative aerobic-anaerobic facultative N. meningitidis. NarE shares structural features and homology with the ADP-ribosylating toxins from V. cholerae and E. coli restricted to residues crucial for transferase activity. When the purifed recombinant NarE reached concentrations of ≥ 7 mg/ml, the protein solution appeared to be golden-brown in colour. UV-visible spectrophotometry and EPR spectroscopy revealed characteristics consistent of an iron-binding protein with structural features resembling those present in the rubredoxin protein family. The presence of iron was determined by colorimetric method and by Atomic Absorption Spectrophotometer. All four cysteine residues, which are the most common iron-ligands, were individually replaced by serine. Substitution of C67 and C128 into serine caused a drastic reduction in the A420/A280 ratio and led to a consistent loss in ADP-ribosyltransferase activity while decrease in NAD-glycohydrolase activity was less dramatic. Moreover, iron affects NarE catalysis on the basis of its different oxidation state. In the presence of the oxidized form of iron (Fe3+) NarE transferase activity is enhanced while the reduced form (Fe2+) is more active on NAD-glycohydrolase activity.