Topological plasticity of enzymes involved in disulfide bond formation allows catalysis in either the periplasm or the cytoplasm

J Mol Biol. 2013 Sep 23;425(18):3268-76. doi: 10.1016/j.jmb.2013.04.034. Epub 2013 Jun 28.


The transmembrane enzymes disulfide bond forming enzyme B (DsbB) and vitamin K epoxide reductase (VKOR) are central to oxidative protein folding in the periplasm of prokaryotes. Catalyzed formation of structural disulfide bonds in proteins also occurs in the cytoplasm of some hyperthermophilic prokaryotes through currently, poorly defined mechanisms. We aimed to determine whether DsbB and VKOR can be inverted in the membrane with retention of activity. By rational design of inversion of membrane topology, we engineered DsbB mutants that catalyze disulfide bond formation in the cytoplasm of Escherichia coli. This represents the first engineered inversion of a transmembrane protein with demonstrated conservation of activity and substrate specificity. This successful designed engineering led us to identify two naturally occurring and oppositely oriented VKOR homologues from the hyperthermophile Aeropyrum pernix that promote oxidative protein folding in the periplasm or cytoplasm, respectively, and hence defines the probable route for disulfide bond formation in the cytoplasm of hyperthermophiles. Our findings demonstrate how knowledge on the determinants of membrane protein topology can be used to de novo engineer a metabolic pathway and to unravel an intriguingly simple evolutionary scenario where a new "adaptive" cellular process is constructed by means of membrane protein topology inversion.

Keywords: DsbB; PDO; TMH; VKOR; protein disulfide oxidoreductase; rational design; topology inversion; transmembrane helix; transmembrane proteins; vitamin K epoxide reductase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aeropyrum / enzymology
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Catalysis
  • Cytoplasm / enzymology*
  • Disulfides / chemistry
  • Disulfides / metabolism*
  • Escherichia coli / enzymology
  • Hydrogen Bonding
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Models, Biological
  • Periplasm / enzymology*
  • Protein Engineering
  • Protein Folding
  • Stereoisomerism
  • Substrate Specificity
  • Vitamin K Epoxide Reductases / chemistry
  • Vitamin K Epoxide Reductases / genetics
  • Vitamin K Epoxide Reductases / metabolism*


  • Bacterial Proteins
  • Disulfides
  • DsbB protein, Bacteria
  • Membrane Proteins
  • Vitamin K Epoxide Reductases