Rho-ADP-ribosylating exoenzyme from Bacillus cereus. Purification, characterization, and identification of the NAD-binding site.
The ADP-ribosyltransferase produced by a pathogenic strain of Bacillus cereus was purified to near homogeneity. The transferase is a 28,000 Da molecular mass enzyme with a pI of 10.3. The specific enzyme activity is 7.0 nmol of ADP-ribose min-1 mg-1 with a Km for NAD of 0.3 microM. Partial amino acid sequence analysis of the exoenzyme reveals no significant homology to Clostridium botulinum C3 nor to Clostridium limosum exoenzyme. The novel exoenzyme selectively modifies the small GTP-binding proteins of the Rho family presumably at the same acceptor amino acid (Asn-41) as determined for C3. Besides cellular Rho, recombinant RhoA and -B are substrates for the exoenzyme. However, recombinant Rac1 and CDC42, although belonging to the Rho family, are not modified. B. cereus exoenzyme was photolabeled with [carbonyl-14C]NAD resulting in inhibition of ADP-ribosyltransferase and NAD-glycohydrolase activity. A glutamic acid residue was identified as part of the NAD-binding site which corresponds to Glu-174 of C3. This glutamic acid is located in a domain which shows high homology with the C-terminal part of C3 exoenzyme, C. limosum exoenzyme, and Staphylococcus aureus EDIN and which probably represents the catalytic site of the transferases. The data indicate that B. cereus exoenzyme is a novel member of the family of C3-like ADP-ribosyltransferases which share the same substrate protein Rho and which have an identical highly conserved catalytic domain.
Just
I
I
Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, Homburg, Germany.
Selzer
J
J
Jung
M
M
van Damme
J
J
Vandekerckhove
J
J
Aktories
K
K
D036002:ADP Ribose Transferases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D001905:Botulinum Toxins; D002384:Catalysis; D003429:Cross Reactions; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D009243:NAD; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins