Abstract
Posttranslational generation of disulfide bonds catalyzed by bacterial Dsb (disulfide bond) enzymes is essential for the oxidative folding of many proteins. Although we now have a good understanding of the Escherichia coli disulfide bond formation system, there are significant gaps in our knowledge concerning the Dsb systems of other bacteria, including Campylobacter jejuni, a food-borne, zoonotic pathogen. We attempted to gain a more complete understanding of the process by thorough analysis of C8J_1298 functioning in vitro and in vivo. C8J_1298 is a homodimeric thiol-oxidoreductase present in wild type (wt) cells, in both reduced and oxidized forms. The protein was previously described as a homolog of DsbC, and thus potentially should be active in rearrangement of disulfides. Indeed, biochemical studies with purified protein revealed that C8J_1298 shares many properties with EcDsbC. However, its activity in vivo is dependent on the genetic background, namely, the set of other Dsb proteins present in the periplasm that determine the redox conditions. In wt C. jejuni cells, C8J_1298 potentially works as a DsbG involved in the control of the cysteine sulfenylation level and protecting single cysteine residues from oxidation to sulfenic acid. A strain lacking only C8J_1298 is indistinguishable from the wild type strain by several assays recognized as the criteria to determine isomerization or oxidative Dsb pathways. Remarkably, in C. jejuni strain lacking DsbA1, the protein involved in generation of disulfides, C8J_1298 acts as an oxidase, similar to the homodimeric oxidoreductase of Helicobater pylori, HP0231. In E. coli, C8J_1298 acts as a bifunctional protein, also resembling HP0231. These findings are strongly supported by phylogenetic data. We also showed that CjDsbD (C8J_0565) is a C8J_1298 redox partner.
Highlights
The disulfide bond formation catalyzed by bacterial proteins of the disulfide bonds (Dsb) system is a crucial step in the folding process of a protein
In silico analysis indicated that C. jejuni encodes many exported proteins containing more than two cysteine residues, potential targets of the oxidative Dsb system
Some proteins with transmembrane helices localized in the cytoplasmic membrane and having periplasmic loops containing cysteine residues may be considered as substrates of the Dsb system
Summary
The disulfide bond formation catalyzed by bacterial proteins of the Dsb (disulfide bond) system is a crucial step in the folding process of a protein. In order to fulfill their roles, most DsbAs cooperate with transmembrane DsbBs that transport electrons into quinone molecules, connecting the disulfide bond generation pathway to the electron transport chain. In this way, DsbBs keep DsbAs in the oxidized form. A disulfide isomerase, acting in Proteus mirabilis cells and atypical in its structure, has recently been characterized It is a trimeric protein whose function is dependent on a shape-shifting motif [5]. Despite differences in their structures, both DsbAs and DsbCs contain similar catalytic domains–a thioredoxin fold with characteristic CXXC and cis-Pro (cP) motifs. Several excellent reviews discussing details of the E. coli Dsb network have recently been published; for example see: [6,7,8,9,10,11,12,13]
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