title: Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase.
abstract: OBJECTIVE
  Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state.
  
  
  RESULTS
  We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG.
  
  
  CONCLUSIONS
  Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization.
authors.affiliation: 1 Protein Expression and Modification, New England Biolabs, Ipswich, Massachusetts.
authors.forename: Claire
authors.initials: C
authors.lastname: Chatelle
authors.affiliation: 1 Protein Expression and Modification, New England Biolabs, Ipswich, Massachusetts.|2 Actelion, Allschwil, Switzerland .
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Kraemer
authors.affiliation: 1 Protein Expression and Modification, New England Biolabs, Ipswich, Massachusetts.
authors.forename: Guoping
authors.initials: G
authors.lastname: Ren
authors.affiliation: 1 Protein Expression and Modification, New England Biolabs, Ipswich, Massachusetts.
authors.forename: Hannah
authors.initials: H
authors.lastname: Chmura
authors.affiliation: 1 Protein Expression and Modification, New England Biolabs, Ipswich, Massachusetts.
authors.forename: Nils
authors.initials: N
authors.lastname: Marechal
authors.affiliation: 3 Department of Microbiology and Immunobiology, Harvard Medical School , Boston, Massachusetts.
authors.forename: Dana
authors.initials: D
authors.lastname: Boyd
authors.affiliation: 1 Protein Expression and Modification, New England Biolabs, Ipswich, Massachusetts.|4 Novartis, Basel, Switzerland .
authors.forename: Caroline
authors.initials: C
authors.lastname: Roggemans
authors.affiliation: 1 Protein Expression and Modification, New England Biolabs, Ipswich, Massachusetts.
authors.forename: Na
authors.initials: N
authors.lastname: Ke
authors.affiliation: 1 Protein Expression and Modification, New England Biolabs, Ipswich, Massachusetts.
authors.forename: Paul
authors.initials: P
authors.lastname: Riggs
authors.affiliation: 5 Howard Hughes Medical Institute Molecular, Cellular and Developmental Biology, University of Michigan , Ann Arbor, Michigan.
authors.forename: James
authors.initials: J
authors.lastname: Bardwell
authors.affiliation: 1 Protein Expression and Modification, New England Biolabs, Ipswich, Massachusetts.
authors.forename: Mehmet
authors.initials: M
authors.lastname: Berkmen
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000596:Amino Acids / Q000378:metabolism; D003300:Copper / Q000633:toxicity; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005820:Genetic Testing; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism; D013434:Sulfenic Acids / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D004220:Disulfides; D013434:Sulfenic Acids; D003300:Copper; D010088:Oxidoreductases; D012259:Ribonuclease, Pancreatic; D000469:Alkaline Phosphatase; D019704:Protein Disulfide-Isomerases