Structure, function, and engineering of disulfide bond isomerization in Escherichia coli
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Bacterial proteins do not contain more than one or two of disulfide bonds in their native structure. As a result, although E. coli periplasm has the means for oxidizing cysteine residues, the cellular folding machinery has not evolved for efficient isomerization of misoxidized disulfide bonds. This issue represents the main limitation in the synthesis of commercially significant multi disulfide-containing heterologous proteins in bacterial cells. In an attempt to engineer E. coli for the expression of recombinant proteins containing multiple disulfides, this work seeks to increase our understanding of the molecular mechanism of bacterial protein disulfide isomerization. This information will in turn facilitate developing protein engineering strategies to tailor the main bacterial isomerase, DsbC, for the enhanced expression of desired heterologous proteins for pharmaceutical or industrial applications. In order to enhance our understanding of the structure-function relationship of DsbC, five putative disulfide isomerases from different bacterial species were identified and their activity characterized in vivo and in vitro. These results were combined with sequence homology analyses and structural information to obtain structure-based interpretation of DsbC function. We showed that the catalytic domain of the DsbC enzyme can be substituted with structurally similar thioredoxin superfamily proteins such as DsbA, and TrxA. These two proteins by themselves catalyze disulfide bond formation and reduction respectively. However, when they are fused to DsbC, substituting for its catalytic domain they catalyze disulfide bond isomerization in a manner similar to the intact DsbC enzyme. Interestingly, some of the DsbC-DsbA protein chimeras were shown to be able to catalyze both protein oxidation and isomerization, two reactions that are kinetically distinct in the cell, and are normally carried out by two separate enzymes. The study of the DsbC-DsbA chimeras pointed to the functional significance of the α-helical linker that serves to join the dimerization domain and the catalytic domain of the protein. To further examine the role of the linker, a series of mutant DsbCs with sequential amino acid deletions in the α-helical linker were constructed. In vivo and in vitro characterization of these mutant enzymes resulted in a better understanding of the role of the linker region in the ability of DsbC to catalyze isomerization while preventing misoxidation of its active site. The above studies identified the key requirements for a bacterial isomerase enzyme as: (a) the presence of substrate binding domain for the interactions with the substrate, (b) the presence of two catalytic domains with thioredoxin fold, and (c) the relative orientation of the active sites in the catalytic domains, determined by the α helical linker. Based on this information we sought to design a molecule capable of catalyzing disulfide isomerization activity, but with no amino acid homology to DsbC. Such a molecule was designed and constructed. The ability of the “artifical disulfide isomerase” to function as DsbC in the periplasm of E. coli, demonstrates that we have in fact identified the functional elements necessary for to the catalysis of disulfide bond isomerization in bacterial cells.