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Aims: The prototypical protein disulfide bond (Dsb) formation and protein refolding pathways in the bacterial periplasm involving Dsb proteins have been most comprehensively defined in Escherichia coli. However, genomic analysis has revealed several distinct Dsb-like systems in bacteria, including the pathogen Salmonella enterica serovar Typhimurium. This includes the scsABCD locus, which encodes a system that has been shown via genetic analysis to confer copper tolerance, but whose biochemical properties at the protein level are not defined. The aim of this study was to provide functional insights into the soluble ScsC protein through structural, biochemical, and genetic analyses., Results: Here we describe the structural and biochemical characterization of ScsC, the soluble DsbA-like component of this system. Our crystal structure of ScsC reveals a similar overall fold to DsbA, although the topology of β-sheets and α-helices in the thioredoxin domains differ. The midpoint reduction potential of the CXXC active site in ScsC was determined to be -132 mV versus normal hydrogen electrode. The reactive site cysteine has a low pKa, typical of the nucleophilic cysteines found in DsbA-like proteins. Deletion of scsC from S. Typhimurium elicits sensitivity to copper (II) ions, suggesting a potential involvement for ScsC in disulfide folding under conditions of copper stress., Innovation and Conclusion: ScsC is a novel disulfide oxidoreductase involved in protection against copper ion toxicity.

Thioredoxin-like proteins of the TlpA/ResE/CcmG subfamily are known to face the periplasm in gram-negative bacteria. Using the tlpA gene of Bradyrhizobium japonicum as a query, we identified a locus (NGO1923) in Neisseria gonorrhoeae that encodes a thioredoxin-like protein (NG_TlpA). Bioinformatics analysis indicated that the predicted NG_TlpA protein contained a cleavable signal peptide at the N terminus, and secondary structure analysis identified a thioredoxin fold with a helical insertion (approximately 25 residues), similar to that found in B. japonicum TlpA but absent in cytoplasmic thioredoxins. Biochemical characterization of a recombinant form of NG_TlpA revealed a standard redox potential (E0') of -206 mV. This property and the observation that the oxidized form of the protein exhibited greater thermal stability than the reduced species indicated that NG_TlpA is a reducing thioredoxin and not an oxidizing thiol-disulfide oxidoreductase like DsbA. The thioredoxin activity of NG_TlpA was confirmed in an insulin disulfide reduction assay. A tlpA mutant of N. gonorrhoeae strain 1291 was found to be highly sensitive to oxidative killing by paraquat and hydrogen peroxide, indicating an antioxidant role for the NG_TlpA in this bacterium. The tlpA mutant also exhibited reduced intracellular survival in human primary cervical epithelial cells.