The nonconsecutive disulfide bond of Escherichia coli phytase (AppA) renders it dependent on the protein-disulfide isomerase, DsbC.
The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane.
Berkmen
Mehmet
M
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Boyd
Dana
D
Beckwith
Jon
J
D010832:6-Phytase / Q000737:chemistry*; D000135:Acid Phosphatase / Q000737:chemistry*; D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D019704:Protein Disulfide-Isomerases / Q000502:physiology*; D017510:Protein Folding
D004220:Disulfides; D029968:Escherichia coli Proteins; C062887:agp protein, E coli; D000135:Acid Phosphatase; D010744:Phosphoric Monoester Hydrolases; D010832:6-Phytase; C406788:appA protein, E coli; D019704:Protein Disulfide-Isomerases