Disulfide Bond Formation and Methionine Sulfoxide Reduction in Streptococcus gordonii

Abstract

Disulfide bonds are important for proper folding and activity of extracytoplasmic proteins. These bonds are formed, reduced, and isomerized by thiol-disulfide oxidoreductases (TDORs). TDORs also catalyze the reduction of methionine sulfoxide to repair oxidatively damaged proteins. A TDOR, named SdbA, which catalyzes disulfide bonds in Streptococcus gordonii, was previously identified. The objectives of this study were to identify the redox partners of SdbA, characterize the methionine sulfoxide reduction pathway, and identify the disulfide bond isomerization pathway in S. gordonii. Using mutational, phenotypic, and biochemical approaches, SdbB and CcdA2 were identified as the redox partners of SdbA. sdbBccdA2 mutants recapitulated the sdbA mutant phenotype and produced inactive AtlS, the natural substrate of SdbA, which lacked a disulfide bond. SdbA was found in a reduced state in the sdbBccdA2 mutant. In S. gordonii, SdbB formed a disulfide-linked complex with SdbA. Using SdbA and SdbB active site variants, we showed that SdbA-SdbB interacts through their N-terminal cysteines. MsrAB was identified as a key enzyme in the methionine sulfoxide reduction pathway with SdbB and another TDOR, Sgo_1177, as immediate redox partners and two membrane proteins, CcdA1 and CcdA2, as downstream partners. The CcdA proteins likely played a role in relaying electrons from the cytoplasm to the pathway. In the cells, MsrAB, SdbB, Sgo_1177, CcdA1, and CcdA2, are needed for protection against oxidative stress. Lastly, SdbB was identified as a potential disulfide bond isomerase with CcdA2 as its redox partner. Both SdbB and CcdA2 are required for the stability and production of a protein with two disulfide bonds and protection against copper stress in S. gordonii. In conclusion, this study advances the understanding of disulfide bond formation and methionine sulfoxide reduction in Gram-positive bacteria. This study gives the first example of a complex oxidative protein-folding pathway in Gram-positive bacteria that consists of an enzyme that uses multiple redox partners to function. It also provides an insight into the extracytoplasmic methionine sulfoxide reduction pathway in S. gordonii. Finally, to the best of my knowledge, this study presents the first evidence that Gram-positive bacteria have a disulfide bond isomerization pathway.