Figure 5

Auto-ADP-ribosylation prefers arginines as target residues. Mutant M8 contains 13 arginines (R) to lysines (K) replacement mutations except Arg530. (A) The same amount of M8 and its catalytically inactive form M8(YEDQ) were incubated with various concentrations of biotinyl-NAD⁺ in the auto-ADP-ribosylation reaction. The arrow indicates the biotinylated bands. (B) Mutant M8 was used as template for the K to R reverse mutation to determine which arginine(s) could be auto-ADP-ribosylated. The arginines present in the M8 and M8 derived mutant enzymes are indicated with their numerical positions in the full-length recombinant cholix toxin shown at the bottom of panel B. (C-E) Variation of acceptor residues were carried out by replacing Lys479 or Lys525 on the mutant M8 with arginine (R), glutamine (Q), asparagine (N), or cysteine (C). The circular dichroism of these M8 derived mutants were shown in (C) and (D). Auto-ADP-ribosylation was performed with wild type or mutant cholix toxin catalytic fragments and detected by Western blotting shown in (E). Arrowheads indicate biotinyl-auto-ADP-ribosylation of the catalytic fragments. Arrows indicate detection of the catalytic fragments by anti-CTc antibody for protein loading control.