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Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA

Lewin, Allison; Crow, Allister; Hodson, Christopher T. C.; Hederstedt, Lars LU and Le Brun, Nick E. (2008) In Biochemical Journal 414. p.81-91
Abstract
The thiol-disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pK(a) values for the CEPC (one-letter code) active-site cysteine residues of Ill are unusual for thioredoxin-like proteins ill that they are both high (> 8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid-base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pK(a) values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, Delta pK(a)... (More)
The thiol-disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pK(a) values for the CEPC (one-letter code) active-site cysteine residues of Ill are unusual for thioredoxin-like proteins ill that they are both high (> 8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid-base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pK(a) values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, Delta pK(a) values of -2.5 were measured for both cysteine residues. Increases in midpoint reduction potentials were also observed, although these were comparatively small: CPHC (DsbA-type) ResA exhibited all increase of +40mV compared with the wild-type protein. Unfolding studies revealed that, despite the observed differences in the properties of the reduced proteins, changes in stability were largely confined to file oxidized state. High-resolution structures of two of the variants (CEHC and CPHC ResA) in their reduced states were determined and are discussed in terms of the observed changes ill properties. Finally, the in vivo functional properties of CEHC RcsA are shown to be significantly affected compared with those of the wild-type protein. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
thioredoxin-like protein, oxidoreductase (TDOR), thiol-disulfide, reduction potential, cytochrome c maturation, pK(a), X-ray crystallography
in
Biochemical Journal
volume
414
pages
81 - 91
publisher
Portland Press Limited
external identifiers
  • wos:000258542100008
  • scopus:49649088871
ISSN
0264-6021
DOI
10.1042/BJ20080356
language
English
LU publication?
yes
id
482e3cfa-3c0f-4520-a8d6-c036a688d478 (old id 1252042)
date added to LUP
2008-11-04 08:33:39
date last changed
2017-05-07 04:02:57
@article{482e3cfa-3c0f-4520-a8d6-c036a688d478,
  abstract     = {The thiol-disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pK(a) values for the CEPC (one-letter code) active-site cysteine residues of Ill are unusual for thioredoxin-like proteins ill that they are both high (> 8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid-base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pK(a) values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, Delta pK(a) values of -2.5 were measured for both cysteine residues. Increases in midpoint reduction potentials were also observed, although these were comparatively small: CPHC (DsbA-type) ResA exhibited all increase of +40mV compared with the wild-type protein. Unfolding studies revealed that, despite the observed differences in the properties of the reduced proteins, changes in stability were largely confined to file oxidized state. High-resolution structures of two of the variants (CEHC and CPHC ResA) in their reduced states were determined and are discussed in terms of the observed changes ill properties. Finally, the in vivo functional properties of CEHC RcsA are shown to be significantly affected compared with those of the wild-type protein.},
  author       = {Lewin, Allison and Crow, Allister and Hodson, Christopher T. C. and Hederstedt, Lars and Le Brun, Nick E.},
  issn         = {0264-6021},
  keyword      = {thioredoxin-like protein,oxidoreductase (TDOR),thiol-disulfide,reduction potential,cytochrome c maturation,pK(a),X-ray crystallography},
  language     = {eng},
  pages        = {81--91},
  publisher    = {Portland Press Limited},
  series       = {Biochemical Journal},
  title        = {Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA},
  url          = {http://dx.doi.org/10.1042/BJ20080356},
  volume       = {414},
  year         = {2008},
}