The putative gamma-glutamylcysteine synthetase from Plasmodium falciparum contains large insertions and a variable...

The tripeptide glutathione plays a pivotal role in the maintenance of the thiol redox state of the cell and for the detoxification of reactive oxygen species. Glutathione is synthesized in two consecutive reactions by y-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase, respectively. The former enzyme represents the rate limiting step of the synthetic pathway. We have cloned the cDNA and gene of a putative gamma-GCS from Plasmodium falciparum. The contiguous cDNA sequences obtained from various cDNA libraries of P. falciparum K1 and 3D7 encompass 4206 bp or 4038 bp and encode polypeptides of 1119 and 1063 amino acids, respectively. The deduced amino acid sequences show four regions of homology (identity: 31.3-43.9%) to human and Trypanosoma brucei gamma-GCS. These regions are interrupted by three large insertions between 94 and 239 amino acids. Within the first insert a variable repetitive motif was identified, which is responsible for the differing sizes of the sequences. We have analysed this phenomenon in five additional P. falciparum strains and found a high degree of variability in the number of the repeated octamer (Y/C)S(N/D)LQQ(Q/R). Therefore the predicted molecular mass of the proteins from different P. falciparum strains ranges from 124.4 to 133.2 kDa, which is almost twice that of the catalytic subunit of the human host enzyme. Isolation of three genomic clones revealed that the gene does not contain introns. P. falciparum gamma-GCS transcription peaks in trophozoites (24-30 h) suggesting that the antioxidant glutathione is predominantly produced at a time where hemoglobin degradation and the simultaneous formation of reactive oxygen species is maximal.