Mono ADP-ribosylation is catalyzed by ADP-ribosyltransferases (mADPRTs) that transfer a single residue of ADP-ribose (ADPR) from NAD to specific amino acids of selected proteins with the simultaneous release of the nicotinamide (nam) moiety. This reaction was initially discovered as the mechanism by which diptheria toxin blocks eukaryotic protein synthesis. A number of other potent bacterial toxins produced by B. pertussis, C. botulinum, E. coli, P. aeruginosa, B. cereus, and V. cholerae were shown to alter host functions by ADP-ribosylating diphtamide, arginine, cysteine and asparagine residues of target proteins involved in critical metabolic or regulatory pathways. Database search in the recently sequenced genome of Neisseria meningitidis (serogroup B, strain MC58) performed using a PSI-BLAST has evidenced an ORF with significant sequence similarity to known prokaryotic and eukaryotic mADPRTs that we named NM-ADPRT. The overall sequence identity with heat-labile enterotoxin from E. Coli is 29.6% and 26.8% with cholera toxin. The homology is mostly restricted to the domains corresponding to the catalytic cleft. In particular the three residues that have been shown to be crucial for ADPRT activity in bacterial toxins like Arg in domain 1 Ser in domain 2 and Glu in domain 3, are also conserved in NM-ADPRT (R7, S70, and E120). Moreover, this transferase shows a biglutamic acid motif typical for arginine specific transferase and NAD-glycohydrolase (E118-X-E12o). To verify whether the NM-ADPRT exerted the predicted enzymatic activities we cloned it in a T7-based plasmid with a His6-tag at the carboxy terminus. The purification of the protein through metal chelate affinity chromatography resulted in a single polypeptide of 20 kDa under SDS-PAGE. This purified NM-ADPRT was shown to utilize the guanidine group of agmatine, a modified arginine, as ADP-ribose acceptor producing ADP-ribosylagmatine at a rate of 6.7± 0.87nmoles hr-1. NM-ADPRT was also able to release nam from NAD at a rate of 8.5 ± 0.35nmoles hr-1 showing NAD-glycohydrolase activity.
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|Titolo:||A NOVEL NAD:ARGININE:ADP-RIBOSYLTRANSFERASE FROM NEISSERIA MENINGITIDIS|
|Data di pubblicazione:||2001|
|Appare nelle tipologie:||Contributo in atto di convegno su volume|