The Glutathione Synthetase of Schizosaccharomyces pombe Is Synthesized as a Homodimer but Retains Full Activity When Present as a Heterotetramer

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. Glutathione (GSH,1 ␥-Glu-Cys-Gly) is a biologically important thiol found in almost all procaryotic and eucaryotic cells. It has been assigned several cellular functions, including protection against oxidative damage, maintenance of a reducing cellular thiol-disulfide balance, electron donation for a number of enzymes, protection of protein sulfhydryls from irreversible oxidation, and detoxification of foreign compounds (1-3). Glutathione is synthesized enzymatically from its constituent amino acids in two consecutive reactions. Glutathione synthetase (EC 6.3.2.3) catalyzes the second step, the addition of glycine to ␥-glutamylcysteine.In most eucaryotic organisms, including Saccharomyces cerevisiae, Arabidopsis thaliana, Homo sapiens, and Rattus norvegicus, glutathione synthetase is a 104 -112-kDa homodimer composed of two identical 52-56-kDa subunits (4 -10). In contrast, the structure of the Schizosaccharomyces pombe enzyme has not been completely elucidated. The enzyme was purified as a 120-kDa heterotetramer consisting of two kinds of subunits, called the "small" and "large" subunits, with apparent molecular masses of 26 and 33 kDa (11). While the gene encoding the 26-kDa subunit was unknown, the gene encoding the 33-kDa subunit, GSH2, had been cloned, and the coding region had been assigned to the 3Ј end (12). When the GSH2 gene was re-sequenced at a later date, however, frameshift errors in the original sequence were identified (13,14). These errors resulted in a shortened open reading frame, downstream from the correct start codon. A protein derived from the full-length GSH2 gene would have a molecular mass of 56 kDa, and BLAST search...

Glutathione Synthesis in Streptococcus agalactiae

“…Glutathione synthesis, catalyzed by the sequential action of ␥-GCS and GS, is nearly ubiquitous in eukaryotes where the tripeptide serves both directly and through enzyme-mediated reactions as an antioxidant and as a sacrificial nucleophile useful in the detoxification of reactive electrophiles (23,27,28). Glutathione synthesis is less common among prokaryotes, but distinct ␥-GCS and GS enzymes have been isolated and characterized from E. coli and several other Gram-negative species (8,19,29,30).…”

Diverse fission yeast genes required for responding to oxidative and metal stress: Comparative analysis of glutathione‐related and other defense gene deletions

“…; Phlippen et al . ). In addition to glutathione and phytochelatin, it was previously reported that the glutathione biosynthetic enzymes could also synthesize ophthalmic acid (γ‐glutamyl‐2‐aminobutyrylglycine) in mouse liver and in cyanobacteria (Orlowski & Wilk ; Soga et al .…”

“…) and that the Δgsa1 mutant requires glutathione supplementation (Phlippen et al . ). Our Δhmt2 data, however, conflict with a previous report in which the JS563 strain (h + ura4‐294 leu1‐32 Δhmt2 ) grew normally on minimal media.…”

“…; Phlippen et al . ), phytochelatin synthase pcs2 + (Clemens et al . ), ubiquinone biosynthetic gene abc1 + (Saiki et al .…”

Site Directed Mutagenesis of Schizosaccharomyces pombe Glutathione Synthetase Produces an Enzyme with Homoglutathione Synthetase Activity

“…This might be due to in vivo protein cleavage as it is known that GSH2 in vitro is cleaved into two parts by a metallo-protease between Ala217 and Ser218 [21] and is active as a hetero tetramer as well as in the non-cleaved homodimeric form. Results of another study revealed that this cleavage might have an influence on the differential substrate recognition in the way that the cleaved enzyme accepts Gly only (unpublished data not shown).…”