RETRACTED ARTICLE: A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo

Abstract

Escherichia coli DsbB is a transmembrane enzyme that catalyzes the re-oxidation of the periplasmic oxidase DsbA by ubiquinone. Here, we sought to convert membrane-bound DsbB into a water-soluble biocatalyst by leveraging a previously described method for in vivo solubilization of integral membrane proteins (IMPs). When solubilized DsbB variants were co-expressed with an export-defective copy of DsbA in the cytoplasm of wild-type E. coli cells, artificial oxidation pathways were created that efficiently catalyzed de novo disulfide bond formation in a range of substrate proteins and in a manner that depended on both DsbA and quinone. Hence, DsbB solubilization was achieved with preservation of both catalytic activity and substrate specificity. Moreover, given the generality of the solubilization technique, the results presented here should pave the way for unlocking the biocatalytic potential of other membrane-bound enzymes whose utility has been limited by poor stability of IMPs outside of their native lipid bilayer context.

Membrane protein engineering to the rescue

“…This allowed the DsbB-DsbA disulphide bond catalysis pathway, which was previously limited to the periplasm, to be fully constituted within the cytoplasm. The modified form of DsbB was still able to interact with DsbA as shown by crosslinking and small angle X-ray scattering (SAXS) studies ( Figure 2B) [32] . The recompartmentalisation of this pathway in the cytoplasm was also enzymatically active, displaying disulphide bond forming capability against several different substrate proteins [32] .…”

“…The modified form of DsbB was still able to interact with DsbA as shown by crosslinking and small angle X-ray scattering (SAXS) studies ( Figure 2B) [32] . The recompartmentalisation of this pathway in the cytoplasm was also enzymatically active, displaying disulphide bond forming capability against several different substrate proteins [32] . Excitingly, this result demonstrates that by engineering IMPs in this way, enzymatic activities previously only associated with lipid membranes, can be transplanted into different cellular compartments and non-native environments without the need for additional solubilising additives.…”

“…The implications of this methodology have wider applications. For instance, the SIMPLEx approach has been used to produce a water soluble form of the bacterial protein DsbB [29,32] . This enzyme, in the presence of ubiquinone, is capable of catalysing the oxidation of the thiodisulphide oxidoreductase partner enzyme DsbA within the bacterial periplasm [33] [34] (Figure 2A).…”

“…DsbB is a membrane bound enzyme which inserts into the inner membrane and thereby limits the oxidising activity of the protein to the periplasm. By producing a water soluble form of DsbB via the addition of the apolipoprotein and decoy fusion tags, this protein can be overexpressed to high levels within the cytoplasm [32] . In that study the partner protein, DsbA, had its periplasmic export sequence removed, meaning it too was retained within the cytoplasm [32] .…”

“…By producing a water soluble form of DsbB via the addition of the apolipoprotein and decoy fusion tags, this protein can be overexpressed to high levels within the cytoplasm [32] . In that study the partner protein, DsbA, had its periplasmic export sequence removed, meaning it too was retained within the cytoplasm [32] . This allowed the DsbB-DsbA disulphide bond catalysis pathway, which was previously limited to the periplasm, to be fully constituted within the cytoplasm.…”