ADP-ribosyltransferase mutations in the catalytic S-1 subunit of pertussis toxin.
The ADP-ribosyltransferase activity of pertussis toxin resides within the S-1 subunit of the toxin. Deletion mapping of a recombinant S-1 subunit produced in Escherichia coli showed that amino acids 2 through 180 are required for ADP-ribosylation of Gi protein. Mutants of the S-1 subunit which lacked either amino acids 2 through 22 or amino acids 153 through 180 failed to express enzyme activity, implicating a functional or structural role for these residues in catalysis. The catalytic carboxy-terminal S-1 deletion, C-180, was found to be more soluble than the recombinant S-1 subunit, making it a useful construct for future structure-function studies on enzyme catalysis. Four independent single-amino-acid substitutions which decreased ADP-ribosyltransferase activity were constructed in the recombinant S-1 subunit. Substitution of Asp-11 by Ser, Arg-13 by Leu, or Trp-26 by Ile decreased enzyme activity to below detectable levels (less than 1.0% of that of the recombinant S-1 subunit). The Glu-139-to-Ser substitution reduced ADP-ribosyltransferase activity to 15% of that of the recombinant S-1 subunit. Both the oxidized and reduced forms of the recombinant S-1 subunit and recombinant S-1 subunits containing single-amino-acid substitutions were degraded through identical immunoreactive tryptic peptides, suggesting that the conformations of the mutants are similar to that of the recombinant S-1 subunit. Identification of noncatalytic forms of the S-1 subunit of pertussis toxin which have conserved protein structure is an initial step in the generation of a recombinant noncatalytic form of pertussis toxin which may be tested as a candidate for an acellular vaccine against Bordetella pertussis.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D019204:GTP-Binding Proteins / Q000378:metabolism; D005796:Genes; D009154:Mutation; D010430:Pentosyltransferases / Q000235:genetics*; D037342:Pertussis Toxin; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000235:genetics*
D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D010430:Pentosyltransferases; D037342:Pertussis Toxin; D014357:Trypsin; D019204:GTP-Binding Proteins