title: Conserved role of the linker alpha-helix of the bacterial disulfide isomerase DsbC in the avoidance of misoxidation by DsbB.
abstract: In the bacterial periplasm the co-existence of a catalyst of disulfide bond formation (DsbA) that is maintained in an oxidized state and of a reduced enzyme that catalyzes the rearrangement of mispaired cysteine residues (DsbC) is important for the folding of proteins containing multiple disulfide bonds. The kinetic partitioning of the DsbA/DsbB and DsbC/DsbD pathways partly depends on the ability of DsbB to oxidize DsbA at rates >1000 times greater than DsbC. We show that the resistance of DsbC to oxidation by DsbB is abolished by deletions of one or more amino acids within the alpha-helix that connects the N-terminal dimerization domain with the C-terminal thioredoxin domain. As a result, mutant DsbC carrying alpha-helix deletions could catalyze disulfide bond formation and complemented the phenotypes of dsbA cells. Examination of DsbC homologues from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Yersinia pseudotuberculosis, Vibrio cholerae (30-70% sequence identity with the Escherichia coli enzyme) revealed that the mechanism responsible for avoiding oxidation by DsbB is a general property of DsbC family enzymes. In addition we found that deletions in the linker region reduced, but did not abolish, the ability of DsbC to assist the formation of active vtPA and phytase in vivo, in a DsbD-dependent manner, revealing that interactions between DsbD and DsbC are also conserved.
authors.affiliation: Department of Chemical Engineering, Institute for Cell and Molecular Biology, University of Texas, Austin, Texas 78712-1095, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Segatori
authors.forename: Lori
authors.initials: L
authors.lastname: Murphy
authors.forename: Silvia
authors.initials: S
authors.lastname: Arredondo
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kadokura
authors.forename: Hiram
authors.initials: H
authors.lastname: Gilbert
authors.forename: Jon
authors.initials: J
authors.lastname: Beckwith
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D002384:Catalysis; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D016974:Dickeya chrysanthemi / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D017353:Gene Deletion; D006193:Haemophilus influenzae / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000502:physiology*; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D033903:Periplasmic Proteins / Q000737:chemistry / Q000502:physiology*; D010641:Phenotype; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity; D013879:Thioredoxins / Q000737:chemistry; D014734:Vibrio cholerae / Q000378:metabolism; D015011:Yersinia pseudotuberculosis / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; C079503:DsbB protein, Bacteria; D029968:Escherichia coli Proteins; D007328:Insulin; D008565:Membrane Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D010088:Oxidoreductases; C109831:DsbG protein, E coli; D003545:Cysteine; D010100:Oxygen