In vitro catalysis of oxidative folding of disulfide-bonded proteins by the Escherichia coli dsbA (ppfA) gene product.
It was shown previously that the Escherichia coli gene ppfA (dsbA) encodes a periplasmic protein, and its inactivation leads to a deficiency in disulfide bond formation of envelope proteins (Kamitani, S., Akiyama, Y., and Ito, K. (1992) EMBO J. 11, 57-62; Bardwell, J. C. A., McGovern, K., and Beckwith, J. (1991) Cell 67, 581-589). The DsbA/PpfA protein was overproduced, purified, and examined for its activities in vitro. Its abundance in a wild-type cell was estimated to be about 850 molecules which probably exist as homodimers as suggested by size exclusion chromatography. Purified DsbA markedly stimulated disulfide bond formation of E. coli alkaline phosphatase, either in vitro synthesized or purified and denatured, as well as of reduced bovine ribonuclease A. The DsbA-catalyzed rapid disulfide bond formation occurred after a lag period which appeared to be determined by the redox state of the reaction mixture and concentration of DsbA. Inclusion of higher concentrations of oxidized glutathione or DsbA shortened the lag period. We propose that DsbA, which proved to directly catalyze disulfide bond formation, may also have a role in maintaining the bacterial periplasm oxidative.
Akiyama
Y
Y
Institute for Virus Research, Kyoto University, Japan.
Kamitani
S
S
Kusukawa
N
N
Ito
K
K
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D007535:Isomerases / Q000378:metabolism*; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004220:Disulfides; D012259:Ribonuclease, Pancreatic; D000469:Alkaline Phosphatase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D005978:Glutathione