The glutathione synthetase of Schizosaccharomyces pombe is synthesized as a homodimer but retains full activity when present as a heterotetramer.
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.
Institute of Biology IV (Microbiology and Genetics), Aachen University, Worringer Weg, D-52056 Aachen, Germany.
Nadine
N
Phlippen
Kurt
K
Hoffmann
Rainer
R
Fischer
Klaus
K
Wolf
Martin
M
Zimmermann
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019143:Evolution, Molecular; D005981:Glutathione Synthase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D005981:Glutathione Synthase