ROLE OF IRON IN CATALYTIC REGULATION OF THE NEISSERIA MENINGITIDIS ADP-RIBOSYLTRANSFERASE ENZYMATIC ACTIVITIES

Among the several toxins used by pathogens to target and kill host cells, proteins that catalyze the ADP-ribosylation reaction represent a family of well characterized enzymes. ADP-ribosylating toxins split the N-glycosidic bond of NAD+ that act as donor of the ADP-ribose moiety, which is selectively linked to specific amino-acids onto protein targets, and nicotinamide, which is simultaneously released. A novel pattern-based computational approach, 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 [1]. NarE retains the ability to ADP-ribosylate arginine and small guanidine compounds like agmatine and to hydrolase NAD in ADP-ribose and nicotinamide (nam) in the absence of ADP-ribose acceptor [2]. A combination of biophysical methods (UV-spectra, EPR) were used to show that NarE contains an iron-sulfur (Fe-S) centre. The native state of the protein shows a complex resonance with g values equal to 4.4, 4.3 and 4.2, indicative of a high spin ferric iron. The iron content of NarE preparation determined by colorimetric assay and by Atomic Absorption Spectrophotometer that converged to a ratio <1-Fe atom per monomer and the structural information obtained by EPR resembled those present in the rubredoxin protein family. Substitution of C67 and C128 into serine caused a reduction in the E420/E280 ratio from 2.3 to 0.72 x 102 indicating the absence of a stable cluster. The mutagenized NarE exerted a consistent decrease of the ADP-ribosyltransferase activity while NAD-glycohydrolase activity remained unaltered [3]. Recent observations indicate that the iron oxidation state exerts a selective regulation of the NarE enzymatic activities. 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. Effects exerted by ferric and ferrous iron were reversed when enzymatic activity were measured in the presence of O-phenanthroline, a potent iron chelator. Therefore the iron through its oxidation state could confer on NarE a new role in Neisseria metabolism.