Photolabeling of Glu-129 of the S-1 subunit of pertussis toxin with NAD.
Infection and immunity
confidence
Key findings
UV irradiation transferred nicotinamide from NAD to Glu-129 of S-1 subunit; replacement with glycine/aspartic acid blocked labeling, implicating Glu-129 as active-site residue.
View source on PubMed (PMID 2807535) ↗
- Population
- In vitro recombinant protein (C180 peptide) containing catalytic region of S-1 subunit of pertussis toxin
- Dosing
- [3H-nicotinamide]NAD and [32P-adenylate]NAD
- Route
- in vitro photolabeling
- Blinding
- not_reported
- Controls
- none
- Drug class
- coenzyme
Full abstract
UV irradiation was shown to induce efficient transfer of radiolabel from nicotinamide-labeled NAD to a recombinant protein (C180 peptide) containing the catalytic region of the S-1 subunit of pertussis toxin. Incorporation of label from [3H-nicotinamide]NAD was efficient (0.5 to 0.6 mol/mol of protein) relative to incorporation from [32P-adenylate]NAD (0.2 mol/mol of protein). Label from [3H-nicotinamide]NAD was specifically associated with Glu-129. Replacement of Glu-129 with glycine or aspartic acid made the protein refractory to photolabeling with [3H-nicotinamide]NAD, whereas replacement of a nearby glutamic acid, Glu-139, with serine did not. Photolabeling of the C180 peptide with NAD is similar to that observed with diphtheria toxin and exotoxin A of Pseudomonas aeruginosa, in which the nicotinamide portion of NAD is transferred to Glu-148 and Glu-553, respectively, in the two toxins. These results implicate Glu-129 of the S-1 subunit as an active-site residue and a potentially important site for genetic modification of pertussis toxin for development of an acellular vaccine against Bordetella pertussis.