Reversion of recombinant toxoids: mutations in diphtheria toxin that partially compensate for active-site deletions.
Deleting an important active-site residue of diphtheria toxin, glutamic acid-148, reduces the toxin's ADP-ribosyltransferase activity by a factor of greater than 10(4). We considered using this mutation to construct a recombinant toxoid for expression by live attenuated vaccines and explored second-site mutations that might cause reversion. Activity was partially restored by substituting glutamic acid for valine-147 or by extending the deletion by five residues toward the NH2 terminus, thereby placing glutamic acid-142 immediately adjacent to tyrosine-149. In both mutants the indicated glutamic acid may occupy a spatial locus similar to that of glutamic acid-148 in the unmutated protein. Simply deleting a crucial residue does not, therefore, provide confidence that a second-site mutation could not readily restore activity to a toxoid.
Killeen
K P
KP
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA.
Escuyer
V
V
Mekalanos
J J
JJ
Collier
R J
RJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D004167:Diphtheria Toxin / Q000235:genetics* / Q000633:toxicity; D004168:Diphtheria Toxoid / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014614:Vaccines, Synthetic / Q000235:genetics
D004167:Diphtheria Toxin; D004168:Diphtheria Toxoid; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D011065:Poly(ADP-ribose) Polymerases