Auto ADP‐ribosylation of NarE, aNeisseria meningitidisADP‐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.

A SIRT4-like auto ADP-ribosyltransferase is essential for the environmental growth of <italic>Mycobacterium smegmatis</italic>

“…Two micrograms of MSMEG_4620 was incubated with 50 mM PIPES or Tris-HCl ( pH 7.2), 150 mM NaCl, and 50 µM biotin-NAD at 25°C for 3 h. After that, the reaction was terminated by boiling for 5 min, and samples were incubated with 0.5 M NaCl, 10 mM HgCl 2 , or 2 M NH 2 OH for additional 2 h at 30°C [13,23]. Finally, samples were subject to western blot analysis as described above.…”

“…Chemical stability assay indicated an arginine-ADP-ribose linkage on MSMEG_4620 ADP-ribosylation usually occurs on arginine, cysteine, and lysine residues [13,23]. To identify the ADP-ribosylated residues of MSMEG_4620, chemical stability experiment was carried out by using HgCl 2 , NH 2 OH, or NaCl that could specifically break the bonds between ADP-ribosyl group and cysteine, arginine, or lysine residue, respectively [14,15,23].…”

“…To identify the ADP-ribosylated residues of MSMEG_4620, chemical stability experiment was carried out by using HgCl 2 , NH 2 OH, or NaCl that could specifically break the bonds between ADP-ribosyl group and cysteine, arginine, or lysine residue, respectively [14,15,23]. The results showed that ADP-ribosyl group could only be removed by NH 2 OH treatment (Fig.…”

“…In addition, NarE, an auto ADP-ribosyltransferase from Neisseria meningitidis, could bind ferric ion through the Fe-S center, which is crucial for its catalytic activity [23]. In order to test whether MSMEG_4620 could be activated by certain metal ions, Ca could also slightly activate it.…”

Opposite Regulatory Effects of Iron Ions on the In Vitro Catalytic Activities of Nare, the Toxin-like ADP-Ribosyltransferase from Neisseria meningitides

“…We have identified an ORF in the N. meningitidis genome that codes for the ADP-ribosyltransferase (ART) NarE, which shares the molecular signature and biochemical features with the ART from V. cholerae and E. coli [10]. In analogy with these two toxins, NarE can ADP-ribosylate arginine, hydrolyze NAD in free ADP-ribose and NAM [11], and ADP-ribosylate itself [12]. A recently discovered feature of NarE is the presence of an iron-sulfur center (Fe-S) that we identified for the first time in a member of the ART family [13].…”