title: Auto ADP-ribosylation of NarE, a Neisseria meningitidis ADP-ribosyltransferase, regulates its catalytic activities.
abstract: 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.
authors: 1Centro Ricerche Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy. enrico.balducci@novartis.com.
authors: Monica
authors: M
authors: Picchianti
authors: Mariangela
authors: M
authors: Del Vecchio
authors: Federica
authors: F
authors: Di Marcello
authors: Massimiliano
authors: M
authors: Biagini
authors: Daniele
authors: D
authors: Veggi
authors: Nathalie
authors: N
authors: Norais
authors: Rino
authors: R
authors: Rappuoli
authors: Mariagrazia
authors: M
authors: Pizza
authors: Enrico
authors: E
authors: Balducci
mesh_terms: D036002:ADP Ribose Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D008969:Molecular Sequence Data; D009154:Mutation; D009244:NAD+ Nucleosidase / Q000378:metabolism; D009345:Neisseria meningitidis / Q000201:enzymology*; D055550:Protein Stability
chemical_list: D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; C480854:NarE protein, Neisseria meningitidis; D009244:NAD+ Nucleosidase
keywords: NAD; NAD-glycohydrolase; pathogenesis