Identification of an Iron-Sulfur Cluster That Modulates the Enzymatic Activity in NarE, a Neisseria meningitidis ADP-ribosyltransferase

• Published in: The Journal of biological chemistry 2009-11, Vol.284 (48), p.33040-33047
• Main Authors: Del Vecchio, Mariangela, Pogni, Rebecca, Baratto, Maria Camilla, Nobbs, Angela, Rappuoli, Rino, Pizza, Mariagrazia, Balducci, Enrico
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate Ribose - metabolism; ADP Ribose Transferases - genetics; ADP Ribose Transferases - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Blotting, Western; Catalysis; Cysteine - genetics; Cysteine - metabolism; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Enzyme Assays; Enzyme Catalysis and Regulation; Escherichia coli; Iron - metabolism; Iron-Sulfur Proteins - genetics; Iron-Sulfur Proteins - metabolism; Mutation; Neisseria meningitidis; Neisseria meningitidis - enzymology; Neisseria meningitidis - genetics; Neisseria meningitidis - metabolism; Niacinamide - metabolism; Recombinant Proteins - metabolism; Serine - genetics; Serine - metabolism; Spectrophotometry; Sulfur - metabolism; Vibrio cholerae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.057547

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 Cys67 and Cys128 into serine caused a drastic reduction in the E420/E280 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.