title: Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin.
abstract: Previous studies of the S1 subunit of pertussis toxin, an NAD(+)-dependent ADP-ribosyltransferase, suggested that a small amino-terminal region of amino acid sequence similarity to the active fragments of both cholera toxin and Escherichia coli heat-labile enterotoxin represents a region containing critical active-site residues that might be involved in the binding of the substrate NAD+. Other studies of two other bacterial toxins possessing ADP-ribosyltransferase activity, diphtheria toxin and Pseudomonas exotoxin A, have revealed the presence of essential glutamic acid residues vicinal to the active site. To help determine the relevance of these observations to activities of the enterotoxins, the A-subunit gene of the E. coli heat-labile enterotoxin was subjected to site-specific mutagenesis in the region encoding the amino-terminal region of similarity to the S1 subunit of pertussis toxin delineated by residues 6 through 17 and at two glutamic acid residues, 110 and 112, that are conserved in the active domains of all of the heat-labile enterotoxin variants and in cholera toxin. Mutant proteins in which arginine 7 was either deleted or replaced with lysine exhibited undetectable levels of ADP-ribosyltransferase activity. However, limited trypsinolysis of the arginine 7 mutants yielded fragmentation kinetics that were different from that yielded by the wild-type recombinant subunit or the authentic A subunit. In contrast, mutant proteins in which glutamic acid residues at either position 110 or 112 were replaced with aspartic acid responded like the wild-type subunit upon limited trypsinolysis, while exhibiting severely depressed, but detectable, ADP-ribosyltransferase activity. The latter results may indicate that either glutamic acid 110 or glutamic acid 112 of the A subunit of heat-labile enterotoxin is analogous to those active-site glutamic acids identified in several other ADP-ribosylating toxins.
authors.affiliation: Laboratory of Vectors and Pathogens, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840.
authors.forename: Y
authors.initials: Y
authors.lastname: Lobet
authors.forename: C W
authors.initials: CW
authors.lastname: Cluff
authors.forename: W
authors.initials: W
authors.lastname: Cieplak
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002417:Cattle; D004269:DNA, Bacterial / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000187:drug effects; D013329:Structure-Activity Relationship
chemical_list: D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D006165:Guanylyl Imidodiphosphate; C027538:heat-labile enterotoxin, E coli; D011065:Poly(ADP-ribose) Polymerases; D004229:Dithiothreitol