Untersuchungen zur Struktur und Funktion der Glutathionsynthetase bei der Spalthefe Schizosaccharomyces pombe
In the literature the enzyme glutathione synthetase of the fission yeast S. pombe had been described - in contrast to the homodimeric enzymes of other eucaryotic organisms - as a heterotetramer composed of two „large” 33 kDa and two „small” 26 kDa subunits. The „large” subunit had been assigned to the 3' region of the GSH2 gene. The sequences of the 56 kDa protein encoded by the full length S. pombe GSH2 open reading frame and glutathione synthetases from other eukaryotes show high levels of homology over the whole alignment. Based on this alignment and on the x-ray coordinates of the human enzyme, a structural model of the fission yeast glutathione synthetase was produced. According to this model, the structure of the fission yeast protein is very similar to its human ortholog. The amino acid residues essential for binding of the substrates and cofactors are highly conserved between the two enzymes. The only striking difference involves a 15 amino acid segment (residues 204 - 218), which only exists in the fission yeast protein. The S. pombe GSH2 gene was cloned, and a histidine-tag was attached to the C-terminus of the protein. The protein was expressed in S. 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 „small” 24 kDa and two „large” 32 kDa subfragments. Both variants showed glutathione synthetase activity. Both the homodimer and the heterotetramer are encoded by the GSH2 gene. The 56 kDa subunit corresponds to the complete GSH2 open reading frame. By MALDI-TOF-MS and peptide mapping, the „small” subfragment was assigned to the 5' area of the open reading frame. The 24 kDa and the 32 kDa proteins are produced following proteolytic cleavage of the 56 kDa protein. The 24 kDa protein represents the N-terminal, the 32 kDa protein the C-terminal subfragment. Protease inhibition experiments showed that the protease responsible for cleaving belongs to the metalloproteases class. The cleavage site is localized between the amino acid residues alanine and serine at positions 217 and 218, as determined by N-terminal sequencing and MALDI-TOF-MS. Site-directed mutagenesis was performed to obtain a stable homodimer: The region around the cleavage site - the amino acid residues 204 - 218 - which only occur in the fission yeast protein, were deleted. The protein was enzymatically active in vivo and in vitro. The regions of the subfragments of glutathione synthetase were subcloned and co-expressed in S. pombe as independent proteins. A heterotetrameric protein, composed of two 24 kDa and 32 kDa subfragments each, was isolated. The protein was functional in vivo and in vitro, which means that the subfragments assemble correctly within the cell. Moreover, a permuted version of the fission yeast glutathione synthetase was created by interchanging the positions of the two subfragments within the protein. The permutation yielded a catalytically active protein. The presence of the additional 15 amino acid residues in the fission yeast protein may trace back to a permutation event during the evolution of glutathione synthetase. The presence of an exon boundary in the respective region of the human gene might indicate the mechanism by which the insert was eliminated during the evolution of metazoans. In a crystallization screen of the S. pombe glutathione synthetase several conditions were identified under which the protein formed glassy aggregates, similar to very small crystals. In order to get bigger monocrystals, suitable for x-ray structure analysis, the conditions will have to be further optimized. As a result, the extraordinary subunit structure of the fission yeast glutathione synthetase was clarified by this work. The theory of a permutation event during the evolution of the enzyme was reproduced experimentally. Furthermore, the homologous expression of several variants of glutathione synthetase showed that S. pombe can serve as a model organism to provide insight into the mechanisms of protein processing and folding within the cell.
info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, Glutathionsynthetase, Glutathion, Molecular Modelling, Proteinfaltung, Proteinevolution, zirkuläre Permutation
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