An in vivo pathway for disulfide bond isomerization in Escherichia coli

Biochemical studies have shown that the periplasmic protein disulfide oxidoreductase DsbC can isomerize aberrant disulfide bonds. Here we present the first evidence for an in vivo role of DsbC in disulfide bond isomerization. Furthermore, our data suggest that the enzymes DsbA and DsbC play distinct roles in the cell in disulfide bond formation and isomerization, respectively. We have shown that mutants in dsbC display a defect in disulfide bond formation specific for proteins with multiple disulfide bonds. The defect can be complemented by the addition of reduced dithiothreitol to the medium, suggesting that absence of DsbC results in accumulation of misoxidized proteins. Mutations in the dipZ and trxA genes have similar phenotypes. We propose that DipZ, a cytoplasmic membrane protein with a thioredoxin-like domain, and thioredoxin, the product of the trxA gene, are components of a pathway for maintaining DsbC active as a protein disulfide bond isomerase.

Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm

Under physiological conditions, the Escherichia coli cytoplasm is maintained in a reduced state that strongly disfavors the formation of stable disulfide bonds in proteins. However, mutants in which the reduction of both thioredoxins and glutathione is impaired (trxB gor mutants) accumulate oxidized, enzymatically active alkaline phosphatase in the cytoplasm. These mutants grow very poorly in the absence of an exogenous reductant and accumulate extragenic suppressors at a high frequency. One such suppressor strain, FA113, grows almost as rapidly as the wild type in the absence of reductant, exhibits slightly faster kinetics of disulfide bond formation, and has fully induced activity of the transcriptional activator, OxyR. FA113 gave substantially higher yields of properly oxidized proteins compared with wild-type or trxB mutant strains. For polypeptides with very complex patterns of disulfide bonds, such as vtPA and the full-length tPA, the amount of active protein was further enhanced up to 15-fold by co-expression of TrxA (thioredoxin 1) mutants with different redox potentials, or 20-fold by the protein disulfide isomerase, DsbC. Remarkably, higher yields of oxidized, biologically active proteins were obtained by expression in the cytoplasm of E. coli FA113 compared with what could be achieved via secretion into the periplasm of a wild-type strain, even under optimized conditions. These results demonstrate that the cytoplasm can be rendered sufficiently oxidizing to allow efficient formation of native disulfide bonds without compromising cell viability.

The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm

Thioredoxin 1 is a major thiol-disulfide oxidoreductase in the cytoplasm of Escherichia coli. One of its functions is presumed to be the reduction of the disulfide bond in the active site of the essential enzyme ribonucleotide reductase. Thioredoxin 1 is kept in a reduced state by thioredoxin reductase. In a thioredoxin reductase null mutant however, most of thioredoxin 1 is in the oxidized form; recent reports have suggested that this oxidized form might promote disulfide bond formation in vivo. In the Escherichia coli periplasm, the protein disulfide isomerase DsbC is maintained in the reduced and active state by the membrane protein DsbD. In a dsbD null mutant, DsbC accumulates in the oxidized form. This oxidized form is then able to promote disulfide bond formation. In both these cases, the inversion of the function of these thiol oxidoreductases appears to be due to an altered redox balance of the environment in which they find themselves. Here, we show that thioredoxin 1 attached to the alkaline phosphatase signal sequence can be exported into the E. coli periplasm. In this new environment for thioredoxin 1, we show that thioredoxin 1 can promote disulfide bond formation and, therefore, partially complement a dsbA strain defective for disulfide bond formation. Thus, we provide evidence that by changing the location of thioredoxin 1 from cytoplasm to periplasm, we change its function from a reductant to an oxidant. We conclude that the in vivo redox function of thioredoxin 1 depends on the redox environment in which it is localized.