The Nonconsecutive Disulfide Bond of Escherichia coli Phytase (AppA) Renders It Dependent on the Protein-disulfide Isomerase, DsbC

Abstract

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.Disulfide bonds between cysteine residues make an important contribution to the folding pathway and stability of many proteins. Early in vitro studies on protein folding showed that the protein RNase when denatured and its disulfide bonds reduced could refold into an active enzyme with the correct disulfide bonds formed (1). Although these results indicated that the information necessary for the proper folding of proteins was intrinsic to the amino acid sequence, the rate of disulfide bond formation in these experiments was much slower than the rates observed in living cells, and the yield of active enzyme was low. The low yields of properly folded enzyme were attributed to the formation of incorrect disulfide bonds in many of the assembled protein molecules. Thus, the in vitro studies, although remarkably successful, were unable to come close to replicating the rapid kinetics and the accuracy of in vivo disulfide bond formation. To explain this inaccuracy observed in biochemical experiments, Anfinsen and co-workers

Nonconsecutive disulfide bond formation in an essential integral outer membrane protein

“…This assumption is based on a model whereby oxidation occurs vectorially while the protein is being translocated across the IM (7). Indeed, such a model has proven correct for PhoA molecules that undergo cotranslational translocation from the cytoplasm (15).…”

“…Introduction of the wrong disulfide bond by DsbA is particularly problematic for proteins that require a disulfide bond between nonconsecutive Cys (7)(8)(9)(10). When such proteins are oxidized incorrectly, the periplasmic disulfide bond isomerase DsbC rearranges their disulfide bonds to produce the final nonconsecutive configuration (11)(12)(13).…”

Conserved Role of the Linker α-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB

“…30 l of the diluted cell lysates were added to 250 l of the same buffer containing 0.04 g of human Glu-type plasminogen (American Diagnostica, Greenwich, CT) per l, and 0.4 mM Spectrozyme PL (American Diagnostica), incubated at 37°C, and the change in A 405 was monitored. Acid phosphatase activities were determined in E. coli MB69 (DHB4 ⌬dsbC) (11) co-transformed with pBAD33 derivatives encoding the DsbC deletion constructs, and pAppA, a pBAD18 derivative encoding the gene appA (11). Cells were grown in LB medium, and enzymatic assays were performed as previously described (11).…”

“…Acid phosphatase activities were determined in E. coli MB69 (DHB4 ⌬dsbC) (11) co-transformed with pBAD33 derivatives encoding the DsbC deletion constructs, and pAppA, a pBAD18 derivative encoding the gene appA (11). Cells were grown in LB medium, and enzymatic assays were performed as previously described (11). To study the in vivo oxidase activity of the proteins, E. coli LM106 (MC1000 dsbA::kan5) and LM102 (MC1000 dsbB::kan5) were transformed with the appropriate pBAD33 plasmid derivatives.…”

Current Progresses in Phytase Research: Three‐Dimensional Structure and Protein Engineering

“…It has been demonstrated that disulfide bonds in AnPhyA and EcAppA are critical for enzymatic activity and protein stability [59,60]. Therefore, introducing additional disulfide bridges is an attractive option for phytase engineering.…”