ACTIVE-SITE MUTATION OF THE C3-LIKE ADP-RIBOSYLTRANSFERASE FROM CLOSTRIDIUM-LIMOSUM - ANALYSIS OF GLUTAMIC-ACID-174

Clostridium limosum ADP-ribosyltransferase modifies low molecular mass GTP-binding proteins of the Rho subtype family. Here we cloned and se quenced the gene of the transferase and expressed it in Escherichia co li. The gene encodes a protein of 250 amino acids (M(r) = 27 840), wit h a putative signal peptide of 45 amino acids, that shows about 60-65% identity with C3 transferases from Clostridium botulinum. The mature C. limosum transferase was expressed as a maltose-binding,r fusion pro tein in E. coil and purified to apparent homogeneity. To study the fun ctional role of Glu 174 of C. limosum transferase, which was recently photoaffinity-labeled with [carbonyl-C-14]NAD [Jung, M., et al. (1993) J. Biol. Chem. 268, 23215-23218], two mutants E174D and E174Q were co nstructed by a polymerase chain reaction-based system. The E174D and E 174Q mutants showed a dramatic decrease in k(cat), but no major change s in K-m,K-NAD. Furthermore, replacement of Glu174 by aspartic acid an d glutamine largely reduced and completely blocked UV-induced incorpor ation of [carbonyl-C-14]NAD into the transferase. The data indicate th at Glu174 is an active site residue of C. limosum transferase.