MINIMUM DOMAIN OF THE SHIGA TOXIN-A SUBUNIT REQUIRED FOR ENZYMATIC-ACTIVITY

The minimum sequence of the enzymatic (A) subunit of Shiga toxin (STX) required for activity was investigated by introducing N-terminal and C-terminal deletions in the molecule. Enzymatic activity was assessed by using an in vitro translation system. A 253-amino-acid STX A polype ptide, which is recognized a the enzymatically active portion of the 2 93-amino-acid A subunit, expressed less than wild-type levels of activ ity. In addition, alteration of the proposed nicking site between Ala- 253 and Ser-254 by site-directed mutagenesis apparently prevented prot eolytic processing but had no effect on the enzymatic activity of the molecule. Therefore, deletion analysis was used to identify amino acid residue 271 as the C terminus of the enzymatically active portion of the STX A subunit. STX A polypeptides with N-terminal and C-terminal d eletions were released into the periplasmic space of Escherichia coli by fusion to the signal peptide and the first 22 amino acids of Shiga- like toxin type II, a member of the STX family. Although these fusion proteins expressed less than wild-type levels of enzymatic activity, t hey confirmed the previous finding that Tyr-77 is an active-site resid ue. Therefore, the minimum domain of the A polypeptide which was requi red for the expression of enzymatic activity was defined as StxA resid ues 75 to 268.