The Rho-deamidating cytotoxic necrotizing factor 1 from Escherichia coli possesses transglutaminase activity. Cysteine 866 and histidine 881 are essential for enzyme activity.
Recently, it has been reported that cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli induces formation of stress fibers by deamidation of glutamine 63 of RhoA (Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., and Aktories, K. (1997) Nature 387, 725-729); Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C., and Boquet, P. (1997) Nature 387, 729-733). By using mass spectrometric analysis, we show now that the toxin transfers ethylenediamine, putrescine, and dansylcadaverine specifically onto glutamine 63 of RhoA. RhoA was also a substrate for guinea pig liver transglutaminase, which modified not only glutamine 63, but also glutamine residues at positions 52 and 136. Treatment of the fully active N-terminal fragment of CNF1 (amino acid residues 709-1014) with iodoacetamide inhibited both deamidation and transglutamination activities. Moreover, exchange of cysteine 866 with serine blocked the enzyme activity of the N-terminal CNF1 fragment. In addition, we identified histidine 881 to be essential for the enzyme activity of CNF1. The data indicate that CNF1 shares a catalytic dyad of cysteine and histidine residues with eukaryotic transglutaminases and cysteine proteases.
Schmidt
G
G
Institut für Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 5, D-79104 Freiburg, Federal Republic of Germany.
Selzer
J
J
Lerm
M
M
Aktories
K
K
D000577:Amides / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006168:Guinea Pigs; D006639:Histidine / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D013329:Structure-Activity Relationship; D011503:Transglutaminases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
D000577:Amides; D001427:Bacterial Toxins; D003603:Cytotoxins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C060296:cytotoxic necrotizing factor type 1; D006639:Histidine; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D003545:Cysteine