Rho-Specific Bacillus cereus ADP-Ribosyltransferase C3cer Cloning and Characterization

Abstract

C3-like ADP-ribosyltransferases represent an expanding family of related exoenzymes, which are produced by Clostridia and various Staphylococcus aureus strains. Here we report on the cloning and biochemical characterization of an ADP-ribosyltransferase from Bacillus cereus strain 2339. The transferase encompasses 219 amino acids; it has a predicted mass of 25.2 kDa and a theoretical isoelectric point of 9.3. To indicate the relationship to the family of C3-like ADP-ribosyltransferases, we termed the enzyme C3cer. The amino acid sequence of C3cer is 30 to 40% identical to other C3-like exoenzymes. By site-directed mutagenesis, Arg(59), Arg(97), Tyr(151), Arg(155), Thr(178), Tyr(180), Gln(183), and Glu(185) of recombinant C3cer were identified as pivotal residues of enzyme activity and/or protein substrate recognition. Precipitation experiments with immobilized RhoA revealed that C3cerTyr(180), which is located in the so-called "ADP-ribosylating toxin turn-turn" (ARTT) motif, plays a major role in the recognition of RhoA. Like other C3-like exoenzymes, C3cer ADP-ribosylates preferentially RhoA and RhoB and to a much lesser extent RhoC. Because the cellular accessibility of recombinant C3cer is low, a fusion toxin (C2IN-C3cer), consisting of the N-terminal 225 amino acid residues of the enzyme component of C2 toxin from Clostridium botulinum and C3cer was used to study the cytotoxic effects of the transferase. This fusion toxin caused rounding up of Vero cells comparable to the effects of Rho-inactivating toxins.

C3 exoenzymes, novel insights into structure and action of Rho-ADP-ribosylating toxins

“…The biochemical analysis supported the essential role of this residue for the enzymatic activity. Exchange of the catalytic glutamate in C3-like ADPribosyltransferases blocked the ADP-ribosylation of RhoA as well as the NAD hydrolysis in the absence of RhoA (Aktories et al 1995;Böhmer et al 1996;Wilde et al 2002bWilde et al , 2003Han et al 2001).…”

“…Exchange of this glutamine residue to alanine or glutamate blocked ADP-ribosylation of Rho; surprisingly, the binding of Rho and NAD and hydrolysis of NAD were hardly affected in these mutant proteins (Ménétrey et al 2002;Wilde et al 2002bWilde et al , 2003. Building on the structural data a role of the glutamine residue in positioning of the acceptor RhoA N41 during formation of the Rho-C3 complex was proposed (Wilde et al 2002b(Wilde et al , 2003. The function of the glutamine residue was analyzed in more detail recently.…”

“…Rho N41 was identified as acceptor amino acid for the ADP-ribosylation of RhoA, B, and C (Sekine et al 1989). Other Rho-family members are poor (Rac for C3bot) or marginal substrates (Cdc42 for C3lim) for C3-like ADP-ribosyltransferases (Just et al 1992;Wilde et al 2003). The highest specificity for RhoA, B, and C was observed with C3cer (Wilde et al 2003).…”

“…Other Rho-family members are poor (Rac for C3bot) or marginal substrates (Cdc42 for C3lim) for C3-like ADP-ribosyltransferases (Just et al 1992;Wilde et al 2003). The highest specificity for RhoA, B, and C was observed with C3cer (Wilde et al 2003). The asparagine modification is a unique property of C3 toxins, since many bacterial ADP-ribosyltransferases modify arginine residues (e.g., cholera toxin, Pseudomonas exoenzyme S and T, C. botulinum C2 toxin; Moss and Vaughan 1977;Barbieri and Sun 2004;Vandekerckhove et al 1988) or a cysteine residue (e.g., pertussis toxin; West et al 1985).…”

Clostridium Botulinum C3 Exoenzyme: Rho-Inactivating Tool in Cell Biology and a Neurotrophic Agent

“…This family further encompasses C3 isoforms from Clostridium limosum (C3 lim ) [20], Bacillus cereus (C3 cer ) [21,22], and Staphylococcus aureus (C3 stau ) [23] ( Table I). The C3-like ADP-ribosyltransferases are secreted by means of a signal peptide and, thus, are classical bacterial exo-enzymes.…”