Enterococcus faecalis glutathione reductase: purification, characterization and expression under normal and hyperbaric O2 conditions

Abstract

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.

Stress Physiology of Lactic Acid Bacteria

“…This relatively large genetic repertoire emphasizes the importance of such enzymes in oxidative stress resistance (for a review, see reference 250). E. faecalis is able to synthesize glutathione and expresses glutathione reductase (261,263).…”

Selenite-stress selected mutant strains of probiotic bacteria for Se source production

“…Similarly, a complete glutathione 325 system against oxidative stress was demonstrated in Lactobacillus 326 fermentum [26]. Selenite resistance induced stress response sys-327 tems that lead up-regulation of GR production In the Gram-positive 328 bacterium E. faecalis, GR has been purified to homogeneity [27]; 329 meanwhile, no GR activity has been described in E. faecium until 330 now. Here, GR activities were detected in E. faecium W54 strain but 331 Table 2 Total glutathione (GSH) and glutathione reductase activity in parental and selenite tolerant mutant bacteria.…”

“…25 Selenium status of people varies by country: intakes are usually 26 high in North America and Japan, varies in China, while lower in 27 Europe (especially in Eastern Europe) and Australia. Low selenium Abbreviations: DTNB, 5,5 -dithobis-(2-nitrobenzoic acid); GR, glutathione reductase; GSH, glutathione; TEA, triethanolamine.…”

Gene cloning and expression analysis of mitochondrial glutathione reductase from Arabian camel (Camelus dromedarius) liver in Escherichia coli

“…The GR obtained in this study exhibited high purity and a subunit molecular weight of approximately 45 kDa; this result is consistent with those of previous studies, which reported wide variation in molecular weights among taxa. Mehul et al (34) reported that GR from Enterococcus faecalis is 51.545 kDa. Bilge et al (35) found that purified GR from rainbow trout is 50 kDa.…”

“…Bilge et al (35) found that purified GR from rainbow trout is 50 kDa. Erat et al (34) purified GR from chicken livers and observed a molecular weight of 43 kDa. William et al (35) showed that GR isolated from the liver of the anoxiatolerant turtle Trachemys scripta elegans is approximately 55 kDa.…”