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Auto ADP‐ribosylation of NarE, a Neisseria meningitidis ADP‐ribosyltransferase, regulates its catalytic activities
Author(s) -
Picchianti Monica,
Del Vecchio Mariangela,
Di Marcello Federica,
Biagini Massimiliano,
Veggi Daniele,
Norais Nathalie,
Rappuoli Rino,
Pizza Mariagrazia,
Balducci Enrico
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.13-229955
Subject(s) - nad+ kinase , adp ribosylation , biochemistry , chemistry , enzyme , biology
NarE is an arginine‐specific mono‐ADP‐ribosyltransferase 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 NAD‐concentration‐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. Site‐directed mutagenesis suggested that the auto‐ADP‐ribosylation site occurred preferentially on the R 7 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. 27, 4723–4730 (2013). www.fasebj.org