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Threonine‐74 Is a Key Site for the Activity of Clostridium perfringens Alpha‐Toxin
Author(s) -
Nagahama Masahiro,
Sakurai Jun
Publication year - 1996
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.1996.tb03333.x
Subject(s) - clostridium perfringens , toxin , biology , microbiology and biotechnology , threonine , alpha (finance) , key (lock) , biochemistry , bacteria , enzyme , ecology , serine , medicine , genetics , construct validity , nursing , patient satisfaction
A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha‐toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha‐toxin. In the mutant toxin, the amino acids at Tyr (Y)‐62, Thr (T)‐74 and Ile (I)‐345 were substituted with His, Ile and Met, respectively. Replacement of T‐74 with Ile by site‐directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250‐fold of that of the wild‐type. The replacement of Y‐62 with Ile or I‐345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [ 65 Zn] 2+ in Tris‐buffered saline, in the same manner as the wild‐type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T‐74 residue plays a key role in these biological activities of C. perfringens alpha‐toxin.