Figure 1.
Multiple amino acid sequence alignment of the GSH synthetases from S. pombe, S. cerevisiae and H. sapiens with the hGSH synthetase sequence of M. trunculata.
SWISSPROT-TREMBL accession numbers are as follows: P35669 (GSH synth. S. pombe), Q08220 (GSH synth. S. cerevisiae), P48637 (GSH synth. human) and AF194422 (hGSH synth. M. trunculata). Dots represent conserved amino acid residues, whereas stars represent identical residues. Ile471, Cys472, Ala485 and Thr486 (fission yeast GSH synthetase) and corresponding residues in the other enzymes are highlighted in grey. Squares – amino acid residues that are part of the ATP binding-site; dotted squares – amino acid residues that are part of the GSH binding site; underlined – amino acids within the entrance of the binding pocket.
Table 1.
Oligonucleotides and vector templates used for site-directed mutagenesis.
Table 2.
Oligonucleotides and vector templates used for site-directed mutagenesis.
Figure 2.
SDS-PAGE analysis of the different mutated forms of the fission yeast GSH synthetase after purification.
Part A shows GSH2-IC/MV, Part B shows GSH-AT/LP and Part C shows GSH2AT/LP-IC/MV. In all cases, the first lane shows the molecular mass standard VIIL with bands of 66, 45, 36, 29, 24, 20 and 14.2 kDa, while the second lane shows the purified GSH2 enzymes (56 kDa).
Table 3.
Enzyme activities and substrate specificities of GSH2 mutants and wild-type GSH synthetase.
Figure 3.
Wireframe surface representation of microenvironment around the amino acid positions (VdW representation) 471, 472, 485 and 486 in the GSH2 wild-type (A) and GSH2-IC/MV-AT/LP (B).
A – GSH2 wild-type: Ile471 (red), Cys472 (yellow), Ala485 (blue) and Thr486 (green); B – GSH2-IC/MV-AT/LP: Met471 (red), Val472 (yello), Leu485 (blue), Pro486 (green).