Biochemical analysis of mutations at tyrosine-98 of the S1 subunit of pertussis toxin.
Molecular modeling and alignment of the primary amino acid sequence of the S1 subunit (S1) of pertussis toxin (PT) with other members of the family of ADP-ribosylating bacterial exotoxins predicted that tyrosine-98 (Y98) of S1 was a conserved residue among these exotoxins. To extend our understanding of the relationship between pertussis toxin and the other ADP-ribosylating exotoxins, we defined the function of Y98 of S1. Using site-directed mutagenesis, Y98 of S1 was substituted with alanine (Y98A), leucine (Y98L), histidine (Y98H), and phenylalanine (Y98F). Mutations were analyzed in the C180 peptide and C219 peptide, recombinant derivatives of S1 which contain the first 180 and 219 amino-terminal residues of S1, respectively. Periplasmic extracts containing the Y98n peptides expressed similar specific activities for the ADP-ribosylation of transducin (Gt) as the periplasmic extract containing wild-type peptides. Mutations at Y98 influenced the subcellular localization of the respective Y98n peptide. The majority of the wild-type Y98 and Y98F peptides localized to the periplasmic extract, while the majority of Y98A and Y98L peptides were associated with the insoluble bacterial outer membrane. Purified C180Y98A and C180Y98F and partially purified C180Y98H peptides possessed similar specific activities for the ADP-ribosylation of Gt as the wild-type C180 peptide. KmNAD and kcat for C180Y98A and C180Y98F in the NAD glycohydrolase reaction were similar to the wild-type C180 peptide. These data show that the R group of Y98 does not participate in the ADP-ribosylation of Gt, but appears to contribute to the proper folding of S1.
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Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Barbieri
J T
JT
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000737:chemistry; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D037342:Pertussis Toxin; D017510:Protein Folding; D013347:Subcellular Fractions / Q000737:chemistry; D013696:Temperature; D015293:Transducin / Q000378:metabolism; D014443:Tyrosine; D010566:Virulence Factors, Bordetella / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D005098:Exotoxins; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D014443:Tyrosine; D037342:Pertussis Toxin; D015293:Transducin