Diphtheria toxin NAD affinity and ADP ribosyltransferase activity are reduced at tryptophan 153 substitutions for alanine or phenylalanine.
Previous studies on chemical modifications of diphtheria toxin (DT) fragment A have suggested that the Trp153 amino acid residue is essential for the ADP ribosylation of elongation factor 2. We verified this experimentally after replacing Trp153 by Phe or Ala residues through in vitro mutagenesis of a cloned toxin gene fragment. Each of the mutant fragment A forms were found to reveal a reduced ADP ribosyl transferase (ADPRT) activity as well as lower affinity for NAD. Both ADPRT activity and NAD affinity of DT fragment A were only partially destroyed by nearly synonymous Trp153 ==> Phe153 substitution, but dramatically destroyed by Ala153 substitution. At the same time, each of the mutant fragment A forms appeared to be thermostable, suggesting that the mutations do not dramatically destroy the structure of the protein. These results clearly demonstrate that Trp153 is not highly specific for DT fragment A structure maintenance, but is highly specific for the key toxin functions such as ADP ribosylation of elongation factor 2 and NAD binding. We suggest that the Trp153 role in DT functioning may be that of binding the ribose moiety of NAD, which is crucial for DT catalytic activity and hence for toxicity.
Zdanovskaia
M V
MV
Laboratory of Biochemical Genetics, Institute for Genetics and Selection of Industrial Microorganisms, Moscow, Russia.
Zdanovsky
A G
AG
Yankovsky
N K
NK
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
D004167:Diphtheria Toxin; D010446:Peptide Fragments; C029795:diphtheria toxin fragment A; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D010649:Phenylalanine; D014364:Tryptophan; D011065:Poly(ADP-ribose) Polymerases; D000409:Alanine