NarE (Neisseria ADP-ribosylating enzyme), identified in the virulent strain MC58 of Nesseria meningitidis, belongs to the family of bacterial ADP-ribosyltransferases [1]. These enzymes catalyze the transfer of the ADP-ribose unit from β-nicotinamide adenine dinucleotide (NAD+) to specific amino-acids (R, C, N, T, Q) with simultaneous release of nicotinamide (nam). In vitro assays showed that NarE is an arginine-specific ADP-ribosyltransferase and also possesses NAD-glycohydrolase (NADase) activity. Recently we showed that NarE contains an iron-sulfur cluster (Fe-S) [2] in which a single iron atom is coordinated with two C (C67 and C128) and two H (H46 and H57) [3]. The presence of a structured cluster is important for transferase activity, which is lost when the cluster is destroyed, while the NADase activity is only slightly decreased. Here we reported that NarE ADP-ribosylates itself in vitro in a dose and concentration dependent manner. Incubation with NH2OH and NaOH hydrolyzed the link between ADP-ribose and NarE suggesting arginine the ADP-ribose acceptor. Four arginine residues (R7, R33, R97, R124) are present in NarE, we mutated each single R to K to identify the modified residue. The mutant R7K does not bind ADP-ribose suggesting that R7 is likely the target of auto-modification. The ADP-ribose unit carries two negative charges and alters considerably the chemical property of the R-residue, which is involved in the binding of NAD and the protein substrate. So the auto-modification influences the NarE enzymatic activities, the ADP-ribosylated NarE decreases transferase activity while NADase proportionally enhances. These data provided evidence that the auto-modification is an intramolecular mechanism adopted by NarE to regulate the priority between the two enzymatic activities. [1] Masignani V. et al Mol. Microbiology (2003) 50(3), 1055-1067 [2] Del Vecchio M. et al. J Biol. Chem. (2009) 284(48):33040-7 [3] Koehler C. et al J Biol. Chem. (2011) in press
IDENTIFICATION OF A MECHANISM FOR ENZYMATIC REGULATION IN NARE
BALDUCCI, Enrico
2011-01-01
Abstract
NarE (Neisseria ADP-ribosylating enzyme), identified in the virulent strain MC58 of Nesseria meningitidis, belongs to the family of bacterial ADP-ribosyltransferases [1]. These enzymes catalyze the transfer of the ADP-ribose unit from β-nicotinamide adenine dinucleotide (NAD+) to specific amino-acids (R, C, N, T, Q) with simultaneous release of nicotinamide (nam). In vitro assays showed that NarE is an arginine-specific ADP-ribosyltransferase and also possesses NAD-glycohydrolase (NADase) activity. Recently we showed that NarE contains an iron-sulfur cluster (Fe-S) [2] in which a single iron atom is coordinated with two C (C67 and C128) and two H (H46 and H57) [3]. The presence of a structured cluster is important for transferase activity, which is lost when the cluster is destroyed, while the NADase activity is only slightly decreased. Here we reported that NarE ADP-ribosylates itself in vitro in a dose and concentration dependent manner. Incubation with NH2OH and NaOH hydrolyzed the link between ADP-ribose and NarE suggesting arginine the ADP-ribose acceptor. Four arginine residues (R7, R33, R97, R124) are present in NarE, we mutated each single R to K to identify the modified residue. The mutant R7K does not bind ADP-ribose suggesting that R7 is likely the target of auto-modification. The ADP-ribose unit carries two negative charges and alters considerably the chemical property of the R-residue, which is involved in the binding of NAD and the protein substrate. So the auto-modification influences the NarE enzymatic activities, the ADP-ribosylated NarE decreases transferase activity while NADase proportionally enhances. These data provided evidence that the auto-modification is an intramolecular mechanism adopted by NarE to regulate the priority between the two enzymatic activities. [1] Masignani V. et al Mol. Microbiology (2003) 50(3), 1055-1067 [2] Del Vecchio M. et al. J Biol. Chem. (2009) 284(48):33040-7 [3] Koehler C. et al J Biol. Chem. (2011) in pressI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
