Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates.
Thioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin ha...
| Main Authors: | , , , , , , , |
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| 格式: | Journal article |
| 語言: | English |
| 出版: |
2008
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| _version_ | 1826276225583153152 |
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| author | Kouwen, T Andréll, J Schrijver, R Dubois, J Maher, M Iwata, S Carpenter, E van Dijl, J |
| author_facet | Kouwen, T Andréll, J Schrijver, R Dubois, J Maher, M Iwata, S Carpenter, E van Dijl, J |
| author_sort | Kouwen, T |
| collection | OXFORD |
| description | Thioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as "mixed disulfide fishing" in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme-substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5A) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes. |
| first_indexed | 2024-03-06T23:10:50Z |
| format | Journal article |
| id | oxford-uuid:65674f77-5bb5-4f55-a0e8-623ab67913c3 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T23:10:50Z |
| publishDate | 2008 |
| record_format | dspace |
| spelling | oxford-uuid:65674f77-5bb5-4f55-a0e8-623ab67913c32022-03-26T18:25:16ZThioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:65674f77-5bb5-4f55-a0e8-623ab67913c3EnglishSymplectic Elements at Oxford2008Kouwen, TAndréll, JSchrijver, RDubois, JMaher, MIwata, SCarpenter, Evan Dijl, JThioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as "mixed disulfide fishing" in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme-substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5A) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes. |
| spellingShingle | Kouwen, T Andréll, J Schrijver, R Dubois, J Maher, M Iwata, S Carpenter, E van Dijl, J Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates. |
| title | Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates. |
| title_full | Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates. |
| title_fullStr | Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates. |
| title_full_unstemmed | Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates. |
| title_short | Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates. |
| title_sort | thioredoxin a active site mutants form mixed disulfide dimers that resemble enzyme substrate reaction intermediates |
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