Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (phospholipase C) which are required for its biological activities.
A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed. Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.
Walker
N
N
Defence Evaluation and Research Agency, Salisbury, Wilts, United Kingdom.
Holley
J
J
Naylor
C E
CE
Flores-Díaz
M
M
Alape-Girón
A
A
Carter
G
G
Carr
F J
FJ
Thelestam
M
M
Keyte
M
M
Moss
D S
DS
Basak
A K
AK
Miller
J
J
Titball
R W
RW
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004530:Egg Yolk / Q000737:chemistry; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006461:Hemolysis; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000378:metabolism; D011487:Protein Conformation; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C033523:4-nitrophenylphosphorylcholine; D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D004269:DNA, Bacterial; D010743:Phospholipids; D010767:Phosphorylcholine; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens; D008239:Lysine; D005998:Glycine