2VOC

THIOREDOXIN A ACTIVE SITE MUTANTS FORM MIXED DISULFIDE DIMERS THAT RESEMBLE ENZYME-SUBSTRATE REACTION INTERMEDIATE

Summary for 2VOC

• Entry DOI: 10.2210/pdb2voc/pdb
• Descriptor: THIOREDOXIN, DI(HYDROXYETHYL)ETHER (3 entities in total)
• Functional Keywords: thioredoxin, thioredoxin fold, electron transport, bacillus, homodimer, disulfide, transport, redox-active center
• Biological source: Bacillus subtilis subsp. subtilis str. 168
• Total number of polymer chains: 2
• Total formula weight: 25024.57

Authors

Kouwen, T.R.H.M.,Andrell, J.,Schrijver, R.,Dubois, J.Y.F.,Maher, M.J.,Iwata, S.,Carpenter, E.P.,van Dijl, J.M. (deposition date: 2008-02-13, release date: 2009-03-10, Last modification date: 2024-10-23)

Primary citation

Kouwen, T.R.,Andrell, J.,Schrijver, R.,Dubois, J.Y.,Maher, M.J.,Iwata, S.,Carpenter, E.P.,van Dijl, J.M.
Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates.
J. Mol. Biol., 379:520-534, 2008

PubMed Abstract: 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.

PubMed: 18455736
DOI: 10.1016/j.jmb.2008.03.077

Experimental method

X-RAY DIFFRACTION (1.5 Å)