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Publication Type
Journal Article
UWI Author(s)
Author, Analytic
Phippen, Nadine; Hoffmann, Kurt; Fischer, Rainer; Wolf, Klaus W.; Zimmermann, Martin
Author Affiliation, Ana.
Electron Microscopy Unit
Article Title
The glutathione synthetase of schizosaccharomyces, pombe is synthetized as a homodimer but retains full activity when present as a hererotetramer
Medium Designator
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Connective Phrase
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Journal Title
Journal of biological chemistry
Translated Title
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Reprint Status
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Date of Publication
2003
Volume ID
278
Issue ID
41
Page(s)
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Language
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Connective Phrase
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Location/URL
http:; www.jbc.org/cgi/reprint/278/41/40152
ISSN
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Notes
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Abstract
Glutathione synthetase was overexpressed as a histidine-tagged protein in Schizosaccharomyces pombe and purified by two-step affinity chromatography. The recovered enzyme occurred in two different forms: a homodimeric protein consisting of two identical 56-kDa subunits and a heterotetrameric protein composed of two 32-kDa and two 24-kDa subfragments. Both forms are encoded by the GSH2 gene. The 56-Da protein corresponds to the complete GSH2 open reading frame, while the subfragments are produced following the cleavage of this larger protein by a metalloprotease. A stable homodimer was obtained by site-directed mutagenesis to remove the protease cleavage site, and this showed normal activity. A structural model of the fission yeast glutathione synthetase was produced, based on the x-ray coordinates of the human enzyme. According to this model the interacting domains of the proteolytic subfragments are strongly entangled. The subfragments were therefore coexpressed as independent proteins. These subfragments assembled correctly to yield functional heterotetramers with equivalent activity to the wild type enzyme. Furthermore, a permuted version of the protein was created. This also showed normal levels of glutathione synthetase activity. These data provide novel insight into the mechanisms of protein folding and the structure and evolution of the glutathione synthetase family.....
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