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Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates.

DOI: 10.1016/j.jmb.2008.03.077 DOI Help
PMID: 18455736 PMID Help

Authors: Thijs R. H. M. Kouwen (University Medical Center Groningen and University of Groningen) , Rianne Schrijver (University Medical Center Groningen and University of Groningen) , Jean-Yves F. Dubois (University Medical Center Groningen and University of Groningen) , Megan J. Maher (Imperial College of Science) , Elizabeth P. Carpenter (Imperial College of Science) , Juni Andrell (Imperial College of Science) , So Iwata (Imperial College of Science) , Jan Maarten Van Dijl (University of Groningen)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Molecular Biology , VOL 379 (3) , PAGES 520-34

State: Published (Approved)
Published: June 2008

Open Access Open Access

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.

Journal Keywords: Bacillus subtilis/chemistry; Bacillus subtilis/metabolism; Bacterial Proteins/metabolism; Binding Sites; Crystallography, X-Ray; Cysteine/chemistry; Dimerization; Hydrogen Bonding; Models, Molecular

Diamond Keywords: Bacteria; Enzymes

Subject Areas: Biology and Bio-materials

Technical Areas:

Added On: 19/08/2009 23:07


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