Enterococcus faecalis glutathione reductase: purification, characterization and expression under normal and hyperbaric O2 conditions.
Glutathione reductase is found ubiquitously in eukaryotes and Gram-negative bacteria, and plays a significant role in bacterial defense against oxidative stress. Glutathione reductase from the Gram-positive bacterium Enterococcus faecalis was purified to homogeneity using anion exchange, hydrophobic interaction, and affinity chromatography. The homogeneous 49-kDa enzyme contained 1 mol bound FAD per subunit. The determined N-terminal amino acid sequence of the E. faecalis enzyme displays significant identity with glutathione reductases from other Gram-negative and Gram-positive bacteria, as well as yeast and human erythrocyte reductases. The kinetic mechanism is ping-pong, and the determined kinetic parameters exhibited by the E. faecalis glutathione reductase are similar to those found for glutathione reductases from yeast, Escherichia coli, and human erythrocyte. A two-fold increased expression of glutathione reductase activity and a three-fold induction of glutathione peroxidase activity were observed under hyperbaric O2 growth conditions without a corresponding change in the total glutathione and soluble thiol content. The difference in the expression of the enzyme, and its cognate substrate's intracellular concentration, under these conditions suggest that the gene encoding glutathione reductase is responsive to oxygen concentration, but that the genes encoding the glutathione synthesizing enzymes are not linked to an oxygen-sensitive promoter.
Patel
M P
MP
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA.
Marcinkeviciene
J
J
Blanchard
J S
JS
D000595:Amino Acid Sequence; D013293:Enterococcus faecalis / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005978:Glutathione / Q000378:metabolism; D005980:Glutathione Reductase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism
D005980:Glutathione Reductase; D005978:Glutathione; D010100:Oxygen