Role of Tryptophan‐1 in Hemolytic and Phospholipase C Activities of Clostridium perfringens Alpha‐Toxin

Abstract

Replacement of the Trp-1 in Clostridium perfringens alpha-toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion. The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of W1G with the metal ion caused little effect on these activities. Gly-Ile-alpha-toxin, which contained an additional Gly-Ile linked to the Nterminal amino acid of alpha-toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild-type toxin. A mutant toxin, which contained an additional Gly-Ile linked to the N-terminus of a protein lacking 4 N-terminal residues of alpha-toxin, showed about 1 and 6% hemolytic and PLC activities of the wild-type toxin, respectively. Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity. These observations suggested that Trp-1 is not essential for toxin activity, but plays a role in binding to zinc ions.

Clostridium sordellii Phospholipase C: Gene Cloning and Comparison of Enzymatic and Biological Activities with Those of Clostridium perfringens and Clostridium bifermentans Phospholipase C

“…The overall charasteristics of clostridial phospholipases, including those of Csp presented here, suggest that phospholipase-producing clostridia possess phospholipases which are similar to each other but unique to the species with respect to structure and function. Nagahama et al (15)(16)(17)(18) and Guillouard et al (7) investigated the roles of amino acid residues of Cpa by site-directed mutagenesis and reported that W1, H68, H126, H136, H148, and E152 were essential for zinc binding, D56 and T74 were required for catalitic activity, and D130 and T272 were required for maintainance of structure. All these residues are Ϫ2 anti-His tag Cpa titer) of His tag Cpa-immunized mice were 6 (n ϭ 2), 9 (n ϭ 2), 10 (n ϭ 1), and 12 (n ϭ 1).…”

Role of the C‐Domain in the Biological Activities of Clostridium perfringens Alpha‐Toxin

“…one zinc ion essential for the catalytic site of the toxin, and the peptide bond between Trp-1 and Asp-2, and the His-11 bind another zinc ion (10,11,14). Kameyama et al (7) reported that the His-148 residue is important for activities of the toxin.…”