BIOCHEMICAL CHARACTERIZATION OF AUTO-ADP-RIBOSYLATED NARE

Mono ADP-ribosylation is a post-translational modification, conserved in viruses, prokaryotes and eukaryotes. Several ADP-ribose transfer enzymes as ART2, ART5, ExoS and CT are able to modify themselves, changing their chemical and biological functions. Here we report that NarE (Neisseria ADP-ribosylating enzyme), a transferase identified in Nesseria meningitides, ADP-ribosylates itself. In vitro ADP-ribosylation assays using NAD as substrate clearly showed the ability of NarE to catalyze an auto-ADP-ribosylation reaction in dose and time dependent manner. Mass analysis further supported the presence of a single ADP-ribose moiety. The ADP-ribose-linkage was destroyed after incubation with hydroxylamine and NaOH indicating that arginine was likely the amino-acid where ADP-ribose was linked. Four arginine residues (R7, R33, R97, R124) are present in NarE sequence, therefore to identify the arginine target we mutated each single R to K. The mutant R7K does not bind ADP-ribose suggesting that R7 is likely the target of auto-modification. R7 is included in Region I, which is involved in NAD binding, therefore, we investigated whether the link of ADP-ribose to R7 alters the interaction with NAD. After auto-ADP-ribosylation NarE shows a reduced ability to bind NAD compared to control experiments. Moreover the auto-ADP-ribosylated protein evidenced a notable change in both its enzymatic activities. The transferase activity is decreased while the NAD-glycohydrolase activity is enanched after modification. So we conclude that the modified NarE is essentially a NAD-glycohydrolase enzyme. Auto ADP-ribosylation to R7 can behave as an intermolecular control mechanism for the enzymatic activities of NarE. Length Max 250 words