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 polypeptide, which is recognized as the enzymatically active portion of the 293-amino-acid A subunit, expressed less than wild-type levels of activity. In addition, alteration of the proposed nicking site between Ala-253 and Ser-254 by site-directed mutagenesis apparently prevented proteolytic 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 deletions 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, they confirmed the previous finding that Tyr-77 is an active-site residue. Therefore, the minimum domain of the A polypeptide which was required for the expression of enzymatic activity was defined as StxA residues 75 to 268.
Haddad
J E
JE
Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan 48201.
al-Jaufy
A Y
AY
Jackson
M P
MP
D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004768:Enterotoxins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D022641:Shiga Toxin 2; D022603:Shiga Toxins; D012762:Shigella dysenteriae / Q000235:genetics*; D013329:Structure-Activity Relationship
D001427:Bacterial Toxins; D004768:Enterotoxins; D011993:Recombinant Fusion Proteins; D022641:Shiga Toxin 2; D022603:Shiga Toxins