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Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA
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Lewin, Allison, Crow, Allister, Hodson, Christopher T. C., Hederstedt, Lars and Le Brun, Nick E. (2008) Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA. Biochemical Journal, 414 (1). pp. 81-91. doi:10.1042/BJ20080356 ISSN 0264-6021.
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Official URL: http://dx.doi.org/10.1042/BJ20080356
Abstract
The thiol–disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pKa values for the CEPC (one-letter code) active-site cysteine residues of ResA are unusual for thioredoxin-like proteins in 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 pKa values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, ΔpKa 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 an increase of +40 mV 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 the 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 in properties. Finally, the in vivo functional properties of CEHC ResA are shown to be significantly affected compared with those of the wild-type protein.
Item Type: | Journal Article | ||||||
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||
Journal or Publication Title: | Biochemical Journal | ||||||
Publisher: | Portland Press | ||||||
ISSN: | 0264-6021 | ||||||
Official Date: | 2008 | ||||||
Dates: |
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Volume: | 414 | ||||||
Number: | 1 | ||||||
Page Range: | pp. 81-91 | ||||||
DOI: | 10.1042/BJ20080356 | ||||||
Status: | Peer Reviewed | ||||||
Publication Status: | Published | ||||||
Access rights to Published version: | Restricted or Subscription Access |
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