Identification of an iron-sulfur cluster that modulates the enzymatic activity in NarE, a Neisseria meningitidis ADP-ribosyltransferase.
In prokaryotes, mono-ADP-ribose transfer enzymes represent a family of exotoxins that display activity in a variety of bacterial pathogens responsible for causing disease in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report here that NarE, a putative ADP-ribosylating toxin previously identified from Neisseria meningitidis, which shares structural homologies with Escherichia coli heat labile enterotoxin and toxin from Vibrio cholerae, possesses an iron-sulfur center. The recombinant protein was expressed in E. coli, and when purified at high concentration, NarE is a distinctive golden brown in color. Evidence from UV-visible spectrophotometry and EPR spectroscopy revealed characteristics consistent of an iron-binding protein. The presence of iron was determined by colorimetric method and by an atomic absorption spectrophotometer. To identify the amino acids involved in binding iron, a combination of site-directed mutagenesis and UV-visible and enzymatic assays were performed. All four cysteine residues were individually replaced by serine. Substitution of Cys(67) and Cys(128) into serine caused a drastic reduction in the E(420)/E(280) ratio, suggesting that these two residues are essential for the formation of a stable coordination. This modification led to a consistent loss in ADP-ribosyltransferase activity, while decrease in NAD-glycohydrolase activity was less dramatic in these mutants, indicating that the correct assembly of the iron-binding site is essential for transferase but not hydrolase activity. This is the first observation suggesting that a member of the ADP-ribosyltransferase family contains an Fe-S cluster implicated in catalysis. This observation may unravel novel functions exerted by this class of enzymes.
Del Vecchio
Mariangela
M
Novartis Vaccines and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy.
Pogni
Rebecca
R
Baratto
Maria Camilla
MC
Nobbs
Angela
A
Rappuoli
Rino
R
Pizza
Mariagrazia
M
Balducci
Enrico
E
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002384:Catalysis; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009345:Neisseria meningitidis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009536:Niacinamide / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D013455:Sulfur / Q000378:metabolism
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D009536:Niacinamide; D012694:Serine; D013455:Sulfur; D007501:Iron; D036002:ADP Ribose Transferases; C480854:NarE protein, Neisseria meningitidis; D003545:Cysteine