The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C-terminal cysteine of the CXXC motif

Mol Microbiol. 2016 Jan;99(2):236-53. doi: 10.1111/mmi.13227. Epub 2015 Oct 30.

Abstract

Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N-terminal cysteine interacts with substrates, whereas the C-terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C(86) P(87) D(88) C(89) catalytic motif. In vitro, SdbA single cysteine variants at the N or C-terminal position (SdbAC86P and SdbAC89A ) were active but displayed different susceptibility to oxidation, and N-terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N-terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C-terminal cysteine alone (in the SdbAC86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C-terminal cysteine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Biocatalysis
  • Catalytic Domain
  • Cysteine / chemistry
  • Cysteine / genetics
  • Cysteine / metabolism*
  • Disulfides / chemistry
  • Disulfides / metabolism*
  • Oxidoreductases / chemistry
  • Oxidoreductases / genetics
  • Oxidoreductases / metabolism*
  • Protein Structure, Tertiary
  • Streptococcus gordonii / chemistry
  • Streptococcus gordonii / enzymology*
  • Streptococcus gordonii / genetics

Substances

  • Bacterial Proteins
  • Disulfides
  • Oxidoreductases
  • Cysteine