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  Kinetic characterization of wild-type and mutant human thioredoxin glutathione reductase defines its reaction and regulatory mechanisms

Brandstaedter, C., Fritz-Wolf, K., Weder, S., Fischer, M., Hecker, B., Rahlfs, S., et al. (2018). Kinetic characterization of wild-type and mutant human thioredoxin glutathione reductase defines its reaction and regulatory mechanisms. The FEBS Journal, 285(3), 542-558. doi:10.1111/febs.14357.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002E-A508-8 Version Permalink: http://hdl.handle.net/21.11116/0000-0007-B64B-8
Genre: Journal Article

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 Creators:
Brandstaedter, Christina, Author
Fritz-Wolf, Karin1, Author              
Weder, Stine, Author
Fischer, Marina, Author
Hecker, Beate, Author
Rahlfs, Stefan, Author
Becker, Katja, Author
Affiliations:
1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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Free keywords: disulfide reductase; flavoproteins; glutaredoxin; redox metabolism; selenoprotein
 Abstract: In most cells, the thioredoxin (Trx) and glutathione systems are essential in maintaining redox homeostasis. The selenoprotein thioredoxin glutathione reductase (TGR) is a hybrid enzyme in which a glutaredoxin (Grx) domain is linked to a thioredoxin reductase (TrxR). Notably, the protein is also capable of reducing glutathione disulfide (GSSG), thus representing an important link between the two redox systems. In this study, we recombinantly produced human TGR (hTGR wild-type) by fusing its open reading frame with a bacterial selenocysteine insertion sequence element and co-expressing the construct in Escherichia coli together with the selA, selB, and selC genes. Additionally, the Sec→Cys mutant (hTGRU642C ) of the full-length protein, the isolated TrxR domain (hTGR151-643 ) and the Grx domain containing a monothiol active site (hTGR1-150 ) were produced and purified. All four proteins were kinetically characterized in direct comparison using Trx, DTNB, HED, or GSSG as the oxidizing substrate. Interestingly, the HED reduction activity was Sec independent and comparable in the full-length protein and the isolated Grx domain, whereas the TrxR and glutathione reductase reactions were clearly selenocysteine dependent, with the GR reaction requiring the Grx domain. Site-directed mutagenesis studies revealed novel insights into the mechanism of GSSG reduction. Furthermore, we identified several glutathionylation sites in hTGR, including Cys93, Cys133, and Cys619, and an inhibitory effect of these modifications on enzyme activity. In contrast to other TGRs, for example, from platyhelminth parasites, hTGR did not exhibit hysteretic behavior. These findings provide new insights into the reaction mechanism and regulation of monothiol Grx-containing TGRs.

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Language(s): eng - English
 Dates: 2017-11-202017-07-312017-12-052017-12-082018-02-01
 Publication Status: Published in print
 Pages: 17
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1111/febs.14357
Other: FEBS14357
Other: 14876322
URI: https://www.ncbi.nlm.nih.gov/pubmed/29222842
 Degree: -

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Title: The FEBS Journal
  Other : The Federation if European Biochemical Societies Journal
Source Genre: Journal
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Publ. Info: Wiley-Blackwell
Pages: - Volume / Issue: 285 (3) Sequence Number: - Start / End Page: 542 - 558 Identifier: ISSN: 1742-464X
CoNE: https://pure.mpg.de/cone/journals/resource/954925398485