NAD-binding site of the C3-like ADP-ribosyltransferase from Clostridium limosum

Treatment of the Rho-ADP-ribosylating C3-like transferase from Clostridium limosum by ultraviolet irradiation in the presence of [carbonyl-14C]NAD incorporated 1 mol of label/mol of exoenzyme. Concomitantly, the transferase and NAD glycohydrolase activity was impaired. A peptide containing the radiolabel was obtained by proteolysis with either staphylococcal protease V8 or trypsin. Their amino acid sequences were Ala/Asp-Gly-Tyr-Ile-Glu-Pro-Ile-Ser-Thr-Phe-Lys-Gly-Gln-Leu-X-Val-Leu-Le u-Pro- Arg and Gly-Gln-Leu-X-Val-Leu-Leu-Pro-Arg, respectively. These sequences correspond with regions Ala-160 through Arg-179 and Gly-171 through Arg-179, respectively, of the very similar Clostridium botulinum C3 transferase, with X being Glu in the unlabeled enzyme. This identifies the glutamic acid residue that corresponds to Glu-174 of C. botulinum C3 transferase as part of the NAD-binding site of the catalytic center of the C. limosum exoenzyme.