Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation.
The Escherichia coli heat-stable enterotoxin STp is synthesized as a precursor consisting of pre, pro and mature regions. Mature STp is released into the culture supernatant and is composed of 18-amino-acid resides which contain three intramolecular disulfide bonds. The involvement of DsbA in the formation of the disulfide bonds of STp was examined in this study. A dsbA mutant was transformed with a plasmid harboring the STp gene, and the ST activity was significantly lower than that of the parent strain harboring the same plasmid. Furthermore, purified DsbA induced the conversion of synthetic STp peptide (inactive form) to the active form and increased the ST activity of the culture supernatant derived from the dsbA transformants. These results showed that DsbA directly catalyzes the formation of the disulfide bonds of STp. DsbA is located in periplasmic space, where STp is released as an intermediate form consisting of pro and mature regions. To examine the effect of the pro region on the action of DsbA, we replaced the cysteine residue at position 39 and tested the effect in vivo. The substitution caused a significant decrease of ST activity in the culture supernatant, the accumulation of inactive ST in periplasmic space, and an alteration in the cleavage site of the intermediate of STp. We conclude that Cys-39 is important for recognition by the processing enzymes required for the maturation of STp.
Yamanaka
H
H
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Japan.
Kameyama
M
M
Baba
T
T
Fujii
Y
Y
Okamoto
K
K
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000378:metabolism*; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007535:Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019704:Protein Disulfide-Isomerases; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship
D001427:Bacterial Toxins; D004220:Disulfides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C034151:heat stable toxin (E coli); D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D003545:Cysteine