NarE is an arginine-specific mono-ADPribosyltransferase
identified in Neisseria meningitidis
that requires the presence of iron in a structured
cluster for its enzymatic activities. In this study, we
show that NarE can perform auto-ADP-ribosylation.
This automodification occurred in a time- and NADconcentration-
dependent manner; was inhibited by novobiocin,
an ADP-ribosyltransferase inhibitor; and did
not occur when NarE was heat inactivated. No reduction
in incorporation was evidenced in the presence of
high concentrations of ATP, GTP, ADP-ribose, or nicotinamide,
which inhibits NAD-glycohydrolase, impeding
the formation of free ADP-ribose. Based on the
electrophoretic profile of NarE on auto-ADP-ribosylation
and on the results of mutagenesis and mass
spectrometry analysis, the auto-ADP-ribosylation appeared
to be restricted to the addition of a single
ADP-ribose. Chemical stability experiments showed
that the ADP-ribosyl linkage was sensitive to hydroxylamine,
which breaks ADP-ribose-arginine bonds. Sitedirected
mutagenesis suggested that the auto-ADP-ribosylation
site occurred preferentially on the R7 residue,
which is located in the region I of the ADP-ribosyltransferase
family. After auto-ADP-ribosylation, NarE
showed a reduction in ADP-ribosyltransferase activity,
while NAD-glycohydrolase activity was increased. Overall,
our findings provide evidence for a novel intramolecular
mechanism used by NarE to regulate its enzymatic
activities.—Picchianti, M., Del Vecchio, M., Di
Marcello, F., Biagini, M., Veggi, D., Norais N., Rappuoli,
R., Pizza, M., Balducci, E. Auto ADP-ribosylation
of NarE, a Neisseria meningitidis ADP-ribosyltransferase,
regulates its catalytic activities. FASEB J