BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF NARE FROM NEISSERIA MENINGITIDIS

BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF NarE FROM Neisseria meningitidis NarE (Neisseria meningitidis ADP-ribosylating enzyme) is an ADP-ribosylating putative toxin identified in the virulent strain MC58 of N. meningitidis serogroup B by computational approach. Methods. NarE was expressed in E. coli as a C-terminal His-tag fusion protein and purified on nickel columns in anaerobic conditions (rNarE). Results. Enzymatic assays have shown that rNarE links the ADP-ribose unit from β-nicotinamide adenine dinucleotide (NAD+) to small guanidine compounds like agmatine at a rate of 6.7±0.87 nmoles hr-1 with the simultaneous release of nicotinamide (Nam). We have noticed that addition of Fe3+ in the mixtures of enzymatic assays enhanced three fold the incorporation of radioactive ADP-ribose, while the presence of o-phenanthroline, a specific iron chelator, drastically reduced the enzymatic activity. These results together with the presence of four Cys spaced as residues of iron-sulfur-cluster motif, prompted us to confirm the presence of this structure using the EPR technique, a method that evidences the presence of paramagnetic ions and identifies the type of coordination in which they are involved. EPR experiments carried out combining rNarE with FeCl3 evidenced the presence of an iron center sourrended by four Cys residues. Further, EPR signal obtained at g=4.3 indicates the presence of a single Fe3+ atom in a distorted sulfur ligands field. In order to confirm the presence of the Fe-S cluster Cys mutated forms of rNarE have already obtained and used for enzymatic assays. Conclusion. Our experiments confirmed, as the rNarE enzymatic activity is strictly dependent by the presence of iron coordinated with the four cysteines