Abstract
Oxidative folding of extracellular proteins is pivotal for the biogenesis of bacterial virulence factors. Escherichia coli DsbA catalyzes disulfide bond formation in extracellular proteins and in multicomponent architectures on the cell surface. The present study assessed the significance of the redox properties of DsbA by exploiting the plaque-forming ability of bacteriophage M13, which specifically recognizes F-pili during infection of the host cell. A library of mutant dsbA genes was constructed by randomizing the dipeptide XX sequence in the active-site redox motif CXXC and then screened for mutants that altered plaque yield and appearance. In total, 24 dsbA mutant alleles produced substantially different degrees of complementation, and one mutant dsbA gene that encodes a CDIC sequence produced over 40-fold more clear plaques than wild type dsbA. The redox potential of purified DsbA [CDIC] was −172 mV, representing a less-oxidizing catalysis than the wild type DsbA (−122 mV), but one that is closer to yeast protein disulfide isomerase (−175 mV). DsbA [CDIC] exhibited a greater ability to refold fully denatured glutathionylated ribonuclease A than the wild type enzyme and a DsbA [CRIC] mutant, which has the same redox potential of −172 mV. Homology modeling and molecular dynamics simulation suggest that the CDIC mutant may have an enlarged substrate-binding cleft near the redox center, which confers kinetic advantages when acting on protein substrates.
| Original language | English |
|---|---|
| Pages (from-to) | 194-201 |
| Number of pages | 8 |
| Journal | Biochimica et Biophysica Acta - Proteins and Proteomics |
| Volume | 1867 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - Mar 1 2019 |
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Keywords
- Disulfide oxidoreductase
- dsbA
- F-pili
- Molecular dynamics
- Redox motif
ASJC Scopus subject areas
- Analytical Chemistry
- Biophysics
- Biochemistry
- Molecular Biology
Cite this
Redox-tuning of oxidizing disulfide oxidoreductase generates a potent disulfide isomerase. / Sutoh, Shinya; Uemura, Yuko; Yamaguchi, Yuko; Kiyotou, Asako; Sugihara, Rena; Nagayasu, Makiko; Kurokawa, Mihoko; Ito, Koreaki; Tsunekawa, Naoki; Nemoto, Michiko; Inagaki, Kenji; Tamura, Takashi.
In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1867, No. 3, 01.03.2019, p. 194-201.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Redox-tuning of oxidizing disulfide oxidoreductase generates a potent disulfide isomerase
AU - Sutoh, Shinya
AU - Uemura, Yuko
AU - Yamaguchi, Yuko
AU - Kiyotou, Asako
AU - Sugihara, Rena
AU - Nagayasu, Makiko
AU - Kurokawa, Mihoko
AU - Ito, Koreaki
AU - Tsunekawa, Naoki
AU - Nemoto, Michiko
AU - Inagaki, Kenji
AU - Tamura, Takashi
PY - 2019/3/1
Y1 - 2019/3/1
N2 - Oxidative folding of extracellular proteins is pivotal for the biogenesis of bacterial virulence factors. Escherichia coli DsbA catalyzes disulfide bond formation in extracellular proteins and in multicomponent architectures on the cell surface. The present study assessed the significance of the redox properties of DsbA by exploiting the plaque-forming ability of bacteriophage M13, which specifically recognizes F-pili during infection of the host cell. A library of mutant dsbA genes was constructed by randomizing the dipeptide XX sequence in the active-site redox motif CXXC and then screened for mutants that altered plaque yield and appearance. In total, 24 dsbA mutant alleles produced substantially different degrees of complementation, and one mutant dsbA gene that encodes a CDIC sequence produced over 40-fold more clear plaques than wild type dsbA. The redox potential of purified DsbA [CDIC] was −172 mV, representing a less-oxidizing catalysis than the wild type DsbA (−122 mV), but one that is closer to yeast protein disulfide isomerase (−175 mV). DsbA [CDIC] exhibited a greater ability to refold fully denatured glutathionylated ribonuclease A than the wild type enzyme and a DsbA [CRIC] mutant, which has the same redox potential of −172 mV. Homology modeling and molecular dynamics simulation suggest that the CDIC mutant may have an enlarged substrate-binding cleft near the redox center, which confers kinetic advantages when acting on protein substrates.
AB - Oxidative folding of extracellular proteins is pivotal for the biogenesis of bacterial virulence factors. Escherichia coli DsbA catalyzes disulfide bond formation in extracellular proteins and in multicomponent architectures on the cell surface. The present study assessed the significance of the redox properties of DsbA by exploiting the plaque-forming ability of bacteriophage M13, which specifically recognizes F-pili during infection of the host cell. A library of mutant dsbA genes was constructed by randomizing the dipeptide XX sequence in the active-site redox motif CXXC and then screened for mutants that altered plaque yield and appearance. In total, 24 dsbA mutant alleles produced substantially different degrees of complementation, and one mutant dsbA gene that encodes a CDIC sequence produced over 40-fold more clear plaques than wild type dsbA. The redox potential of purified DsbA [CDIC] was −172 mV, representing a less-oxidizing catalysis than the wild type DsbA (−122 mV), but one that is closer to yeast protein disulfide isomerase (−175 mV). DsbA [CDIC] exhibited a greater ability to refold fully denatured glutathionylated ribonuclease A than the wild type enzyme and a DsbA [CRIC] mutant, which has the same redox potential of −172 mV. Homology modeling and molecular dynamics simulation suggest that the CDIC mutant may have an enlarged substrate-binding cleft near the redox center, which confers kinetic advantages when acting on protein substrates.
KW - Disulfide oxidoreductase
KW - dsbA
KW - F-pili
KW - Molecular dynamics
KW - Redox motif
UR - http://www.scopus.com/inward/record.url?scp=85059038211&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85059038211&partnerID=8YFLogxK
U2 - 10.1016/j.bbapap.2018.12.005
DO - 10.1016/j.bbapap.2018.12.005
M3 - Article
VL - 1867
SP - 194
EP - 201
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
SN - 1570-9639
IS - 3
ER -